GABA RECEPTOR

DR NITIN SHINDEFYR DEPARTMENT OF PHARMACOLOGYLokmanya Tilak Municipal Medical College amp GH SION MUMBAI-22DATE11-01-2014

CONTENTS

Section-A (Basic Understanding Of GABA Receptors And Their Types)

1 Neurotransmitter - GABA

2 GABA SYNTHESIS UPTAKE AND METABOLISM 3 ABC hellip types of GABA receptors4 GABA A receptor

a) GABA ndashA- structureb) GABA ndashA- subunitsc) GABA ndashA- MOA

5 Extra Synaptic GABA A receptors 6 GABA B receptorsa) Introductionb) Molecular structure of GABA B receptorc) MOA of GABA B receptor

Neurotransmitter - GABA

4-aminobutanoic acid

bull Chief inhibitory neurotransmitter in mammalian central nervous system

bull Approximately 40 neurons in mamalian CNS are purely gabaminergic

Synthesis of GABA

GABA SYNTHESIS UPTAKE AND METABOLISM

Synthesized in brain ndash GABA shunt

GABA SYNTHESIS UPTAKE AND METABOLISM

ABChelliphellipTypes of GABA Receptorbull Three main types A B and C

bull The GABAA receptor is a ligand-gated Clndash ion channel an ionotropic receptorldquo

bull The GABAB receptor is a GPCRmetabotropic

bull The GABAC receptor is a transmitter-gated Clndash channelNewly discovered

Subunits at GABA ndash A

SUBUNITS OF GABA

GABAA receptor structure

bull It is a multimeric transmembrane receptor that consists of 5 subunits arranged around a central Clminus ion pore

bull Members of family of Cys-loop ligand-gated ion channels (loop formed by a disulfide bond betn 2 cysteine residues)

bull The receptor is usually located postsynaptically However some isoforms may be found extrasynaptically

bull Each subunit is composed of a polypeptide sequence of approximately 450ndash630 amino acids (40ndash60 kDa) with large N-terminal and smaller C-terminal extracellular domains

bull individual subunits contain four distinct transmembrane(TM) domains with the second transmembrane domain (TM2) lining the channel lumen

bull A large intracellular loop connects the TM3 and TM4 regions providing sites for phosphorylationby a range of serine threonine aspargine amp tyrosine kinases

GABAA receptor MOA

bull Binding of GABA triggers opening of the Clminus ion pore

bull This drives the membrane potential towards the reversal potential of the Clmacr ion which is about ndash65 mV in neurons inhibiting the firing of new action potentials

bull This makes it more difficult for excitatory neurotransmitters to depolarize the neuron

bull The net effect is typically inhibitory reducing the activity of the neuron

Video clip

>

Extrasynaptic GABAA Receptorsbull Synaptic GABAA receptors Underlie

ldquophasicrdquo inhibitionbull By contrast extra-synaptic receptors

are usually exposed to low but persistent

GABA concentrations leading to ldquotonicrdquo inhibition

bull Compared to their synaptic counterparts these showi Increased sensitivity to GABAii Reduced propensity to desensitizeiii More rapid deactivation phase after removal of GABA

Extrasynaptic GABAA Receptors

bull The occurrence of tonic GABAA inhibition coincides with the expression of relatively rare receptor subunits articularly the α4 α6 and δ subunits and as a general rule-of-thumb δ subunit-containing receptors are extrasynaptic

bull The subunit can also govern receptor pharmacologyndash extrasynaptic GABAARs are insensitive to benzodiazepine

agonists ndash but highly sensitive to the GABAAR ldquosuper agonistrdquo

tetrahydroisoxazolopyridineol (THIPgaboxadol)

Extrasynaptic GABAA Receptorsamp drugs

Studies have begun to identify extrasynaptic GABAARs as novel targets for a diverse array of endogenous and

clinically relevant agents including

bull Certain Neuroactive steroidsbull Amino acid Taurinebull Ethanolbull Several Anestheticbull Hypnotic agentsbull Analgesicsbull Some Anticonvulsant drugs

SUBUNIT AND PHARMACOLOGICAL ACTION

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

CONTENTS

Section-A (Basic Understanding Of GABA Receptors And Their Types)

1 Neurotransmitter - GABA

2 GABA SYNTHESIS UPTAKE AND METABOLISM 3 ABC hellip types of GABA receptors4 GABA A receptor

a) GABA ndashA- structureb) GABA ndashA- subunitsc) GABA ndashA- MOA

5 Extra Synaptic GABA A receptors 6 GABA B receptorsa) Introductionb) Molecular structure of GABA B receptorc) MOA of GABA B receptor

Neurotransmitter - GABA

4-aminobutanoic acid

bull Chief inhibitory neurotransmitter in mammalian central nervous system

bull Approximately 40 neurons in mamalian CNS are purely gabaminergic

Synthesis of GABA

GABA SYNTHESIS UPTAKE AND METABOLISM

Synthesized in brain ndash GABA shunt

GABA SYNTHESIS UPTAKE AND METABOLISM

ABChelliphellipTypes of GABA Receptorbull Three main types A B and C

bull The GABAA receptor is a ligand-gated Clndash ion channel an ionotropic receptorldquo

bull The GABAB receptor is a GPCRmetabotropic

bull The GABAC receptor is a transmitter-gated Clndash channelNewly discovered

Subunits at GABA ndash A

SUBUNITS OF GABA

GABAA receptor structure

bull It is a multimeric transmembrane receptor that consists of 5 subunits arranged around a central Clminus ion pore

bull Members of family of Cys-loop ligand-gated ion channels (loop formed by a disulfide bond betn 2 cysteine residues)

bull The receptor is usually located postsynaptically However some isoforms may be found extrasynaptically

bull Each subunit is composed of a polypeptide sequence of approximately 450ndash630 amino acids (40ndash60 kDa) with large N-terminal and smaller C-terminal extracellular domains

bull individual subunits contain four distinct transmembrane(TM) domains with the second transmembrane domain (TM2) lining the channel lumen

bull A large intracellular loop connects the TM3 and TM4 regions providing sites for phosphorylationby a range of serine threonine aspargine amp tyrosine kinases

GABAA receptor MOA

bull Binding of GABA triggers opening of the Clminus ion pore

bull This drives the membrane potential towards the reversal potential of the Clmacr ion which is about ndash65 mV in neurons inhibiting the firing of new action potentials

bull This makes it more difficult for excitatory neurotransmitters to depolarize the neuron

bull The net effect is typically inhibitory reducing the activity of the neuron

Video clip

>

Extrasynaptic GABAA Receptorsbull Synaptic GABAA receptors Underlie

ldquophasicrdquo inhibitionbull By contrast extra-synaptic receptors

are usually exposed to low but persistent

GABA concentrations leading to ldquotonicrdquo inhibition

bull Compared to their synaptic counterparts these showi Increased sensitivity to GABAii Reduced propensity to desensitizeiii More rapid deactivation phase after removal of GABA

Extrasynaptic GABAA Receptors

bull The occurrence of tonic GABAA inhibition coincides with the expression of relatively rare receptor subunits articularly the α4 α6 and δ subunits and as a general rule-of-thumb δ subunit-containing receptors are extrasynaptic

bull The subunit can also govern receptor pharmacologyndash extrasynaptic GABAARs are insensitive to benzodiazepine

agonists ndash but highly sensitive to the GABAAR ldquosuper agonistrdquo

tetrahydroisoxazolopyridineol (THIPgaboxadol)

Extrasynaptic GABAA Receptorsamp drugs

Studies have begun to identify extrasynaptic GABAARs as novel targets for a diverse array of endogenous and

clinically relevant agents including

bull Certain Neuroactive steroidsbull Amino acid Taurinebull Ethanolbull Several Anestheticbull Hypnotic agentsbull Analgesicsbull Some Anticonvulsant drugs

SUBUNIT AND PHARMACOLOGICAL ACTION

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Neurotransmitter - GABA

4-aminobutanoic acid

bull Chief inhibitory neurotransmitter in mammalian central nervous system

bull Approximately 40 neurons in mamalian CNS are purely gabaminergic

Synthesis of GABA

GABA SYNTHESIS UPTAKE AND METABOLISM

Synthesized in brain ndash GABA shunt

GABA SYNTHESIS UPTAKE AND METABOLISM

ABChelliphellipTypes of GABA Receptorbull Three main types A B and C

bull The GABAA receptor is a ligand-gated Clndash ion channel an ionotropic receptorldquo

bull The GABAB receptor is a GPCRmetabotropic

bull The GABAC receptor is a transmitter-gated Clndash channelNewly discovered

Subunits at GABA ndash A

SUBUNITS OF GABA

GABAA receptor structure

bull It is a multimeric transmembrane receptor that consists of 5 subunits arranged around a central Clminus ion pore

bull Members of family of Cys-loop ligand-gated ion channels (loop formed by a disulfide bond betn 2 cysteine residues)

bull The receptor is usually located postsynaptically However some isoforms may be found extrasynaptically

bull Each subunit is composed of a polypeptide sequence of approximately 450ndash630 amino acids (40ndash60 kDa) with large N-terminal and smaller C-terminal extracellular domains

bull individual subunits contain four distinct transmembrane(TM) domains with the second transmembrane domain (TM2) lining the channel lumen

bull A large intracellular loop connects the TM3 and TM4 regions providing sites for phosphorylationby a range of serine threonine aspargine amp tyrosine kinases

GABAA receptor MOA

bull Binding of GABA triggers opening of the Clminus ion pore

bull This drives the membrane potential towards the reversal potential of the Clmacr ion which is about ndash65 mV in neurons inhibiting the firing of new action potentials

bull This makes it more difficult for excitatory neurotransmitters to depolarize the neuron

bull The net effect is typically inhibitory reducing the activity of the neuron

Video clip

>

Extrasynaptic GABAA Receptorsbull Synaptic GABAA receptors Underlie

ldquophasicrdquo inhibitionbull By contrast extra-synaptic receptors

are usually exposed to low but persistent

GABA concentrations leading to ldquotonicrdquo inhibition

bull Compared to their synaptic counterparts these showi Increased sensitivity to GABAii Reduced propensity to desensitizeiii More rapid deactivation phase after removal of GABA

Extrasynaptic GABAA Receptors

bull The occurrence of tonic GABAA inhibition coincides with the expression of relatively rare receptor subunits articularly the α4 α6 and δ subunits and as a general rule-of-thumb δ subunit-containing receptors are extrasynaptic

bull The subunit can also govern receptor pharmacologyndash extrasynaptic GABAARs are insensitive to benzodiazepine

agonists ndash but highly sensitive to the GABAAR ldquosuper agonistrdquo

tetrahydroisoxazolopyridineol (THIPgaboxadol)

Extrasynaptic GABAA Receptorsamp drugs

Studies have begun to identify extrasynaptic GABAARs as novel targets for a diverse array of endogenous and

clinically relevant agents including

bull Certain Neuroactive steroidsbull Amino acid Taurinebull Ethanolbull Several Anestheticbull Hypnotic agentsbull Analgesicsbull Some Anticonvulsant drugs

SUBUNIT AND PHARMACOLOGICAL ACTION

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

GABA SYNTHESIS UPTAKE AND METABOLISM

Synthesized in brain ndash GABA shunt

GABA SYNTHESIS UPTAKE AND METABOLISM

ABChelliphellipTypes of GABA Receptorbull Three main types A B and C

bull The GABAA receptor is a ligand-gated Clndash ion channel an ionotropic receptorldquo

bull The GABAB receptor is a GPCRmetabotropic

bull The GABAC receptor is a transmitter-gated Clndash channelNewly discovered

Subunits at GABA ndash A

SUBUNITS OF GABA

GABAA receptor structure

bull It is a multimeric transmembrane receptor that consists of 5 subunits arranged around a central Clminus ion pore

bull Members of family of Cys-loop ligand-gated ion channels (loop formed by a disulfide bond betn 2 cysteine residues)

bull The receptor is usually located postsynaptically However some isoforms may be found extrasynaptically

bull Each subunit is composed of a polypeptide sequence of approximately 450ndash630 amino acids (40ndash60 kDa) with large N-terminal and smaller C-terminal extracellular domains

bull individual subunits contain four distinct transmembrane(TM) domains with the second transmembrane domain (TM2) lining the channel lumen

bull A large intracellular loop connects the TM3 and TM4 regions providing sites for phosphorylationby a range of serine threonine aspargine amp tyrosine kinases

GABAA receptor MOA

bull Binding of GABA triggers opening of the Clminus ion pore

bull This drives the membrane potential towards the reversal potential of the Clmacr ion which is about ndash65 mV in neurons inhibiting the firing of new action potentials

bull This makes it more difficult for excitatory neurotransmitters to depolarize the neuron

bull The net effect is typically inhibitory reducing the activity of the neuron

Video clip

>

Extrasynaptic GABAA Receptorsbull Synaptic GABAA receptors Underlie

ldquophasicrdquo inhibitionbull By contrast extra-synaptic receptors

are usually exposed to low but persistent

GABA concentrations leading to ldquotonicrdquo inhibition

bull Compared to their synaptic counterparts these showi Increased sensitivity to GABAii Reduced propensity to desensitizeiii More rapid deactivation phase after removal of GABA

Extrasynaptic GABAA Receptors

bull The occurrence of tonic GABAA inhibition coincides with the expression of relatively rare receptor subunits articularly the α4 α6 and δ subunits and as a general rule-of-thumb δ subunit-containing receptors are extrasynaptic

bull The subunit can also govern receptor pharmacologyndash extrasynaptic GABAARs are insensitive to benzodiazepine

agonists ndash but highly sensitive to the GABAAR ldquosuper agonistrdquo

tetrahydroisoxazolopyridineol (THIPgaboxadol)

Extrasynaptic GABAA Receptorsamp drugs

Studies have begun to identify extrasynaptic GABAARs as novel targets for a diverse array of endogenous and

clinically relevant agents including

bull Certain Neuroactive steroidsbull Amino acid Taurinebull Ethanolbull Several Anestheticbull Hypnotic agentsbull Analgesicsbull Some Anticonvulsant drugs

SUBUNIT AND PHARMACOLOGICAL ACTION

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

GABA SYNTHESIS UPTAKE AND METABOLISM

ABChelliphellipTypes of GABA Receptorbull Three main types A B and C

bull The GABAA receptor is a ligand-gated Clndash ion channel an ionotropic receptorldquo

bull The GABAB receptor is a GPCRmetabotropic

bull The GABAC receptor is a transmitter-gated Clndash channelNewly discovered

Subunits at GABA ndash A

SUBUNITS OF GABA

GABAA receptor structure

bull It is a multimeric transmembrane receptor that consists of 5 subunits arranged around a central Clminus ion pore

bull Members of family of Cys-loop ligand-gated ion channels (loop formed by a disulfide bond betn 2 cysteine residues)

bull The receptor is usually located postsynaptically However some isoforms may be found extrasynaptically

bull Each subunit is composed of a polypeptide sequence of approximately 450ndash630 amino acids (40ndash60 kDa) with large N-terminal and smaller C-terminal extracellular domains

bull individual subunits contain four distinct transmembrane(TM) domains with the second transmembrane domain (TM2) lining the channel lumen

bull A large intracellular loop connects the TM3 and TM4 regions providing sites for phosphorylationby a range of serine threonine aspargine amp tyrosine kinases

GABAA receptor MOA

bull Binding of GABA triggers opening of the Clminus ion pore

bull This drives the membrane potential towards the reversal potential of the Clmacr ion which is about ndash65 mV in neurons inhibiting the firing of new action potentials

bull This makes it more difficult for excitatory neurotransmitters to depolarize the neuron

bull The net effect is typically inhibitory reducing the activity of the neuron

Video clip

>

Extrasynaptic GABAA Receptorsbull Synaptic GABAA receptors Underlie

ldquophasicrdquo inhibitionbull By contrast extra-synaptic receptors

are usually exposed to low but persistent

GABA concentrations leading to ldquotonicrdquo inhibition

bull Compared to their synaptic counterparts these showi Increased sensitivity to GABAii Reduced propensity to desensitizeiii More rapid deactivation phase after removal of GABA

Extrasynaptic GABAA Receptors

bull The occurrence of tonic GABAA inhibition coincides with the expression of relatively rare receptor subunits articularly the α4 α6 and δ subunits and as a general rule-of-thumb δ subunit-containing receptors are extrasynaptic

bull The subunit can also govern receptor pharmacologyndash extrasynaptic GABAARs are insensitive to benzodiazepine

agonists ndash but highly sensitive to the GABAAR ldquosuper agonistrdquo

tetrahydroisoxazolopyridineol (THIPgaboxadol)

Extrasynaptic GABAA Receptorsamp drugs

Studies have begun to identify extrasynaptic GABAARs as novel targets for a diverse array of endogenous and

clinically relevant agents including

bull Certain Neuroactive steroidsbull Amino acid Taurinebull Ethanolbull Several Anestheticbull Hypnotic agentsbull Analgesicsbull Some Anticonvulsant drugs

SUBUNIT AND PHARMACOLOGICAL ACTION

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

ABChelliphellipTypes of GABA Receptorbull Three main types A B and C

bull The GABAA receptor is a ligand-gated Clndash ion channel an ionotropic receptorldquo

bull The GABAB receptor is a GPCRmetabotropic

bull The GABAC receptor is a transmitter-gated Clndash channelNewly discovered

Subunits at GABA ndash A

SUBUNITS OF GABA

GABAA receptor structure

bull It is a multimeric transmembrane receptor that consists of 5 subunits arranged around a central Clminus ion pore

bull Members of family of Cys-loop ligand-gated ion channels (loop formed by a disulfide bond betn 2 cysteine residues)

bull The receptor is usually located postsynaptically However some isoforms may be found extrasynaptically

bull Each subunit is composed of a polypeptide sequence of approximately 450ndash630 amino acids (40ndash60 kDa) with large N-terminal and smaller C-terminal extracellular domains

bull individual subunits contain four distinct transmembrane(TM) domains with the second transmembrane domain (TM2) lining the channel lumen

bull A large intracellular loop connects the TM3 and TM4 regions providing sites for phosphorylationby a range of serine threonine aspargine amp tyrosine kinases

GABAA receptor MOA

bull Binding of GABA triggers opening of the Clminus ion pore

bull This drives the membrane potential towards the reversal potential of the Clmacr ion which is about ndash65 mV in neurons inhibiting the firing of new action potentials

bull This makes it more difficult for excitatory neurotransmitters to depolarize the neuron

bull The net effect is typically inhibitory reducing the activity of the neuron

Video clip

>

Extrasynaptic GABAA Receptorsbull Synaptic GABAA receptors Underlie

ldquophasicrdquo inhibitionbull By contrast extra-synaptic receptors

are usually exposed to low but persistent

GABA concentrations leading to ldquotonicrdquo inhibition

bull Compared to their synaptic counterparts these showi Increased sensitivity to GABAii Reduced propensity to desensitizeiii More rapid deactivation phase after removal of GABA

Extrasynaptic GABAA Receptors

bull The occurrence of tonic GABAA inhibition coincides with the expression of relatively rare receptor subunits articularly the α4 α6 and δ subunits and as a general rule-of-thumb δ subunit-containing receptors are extrasynaptic

bull The subunit can also govern receptor pharmacologyndash extrasynaptic GABAARs are insensitive to benzodiazepine

agonists ndash but highly sensitive to the GABAAR ldquosuper agonistrdquo

tetrahydroisoxazolopyridineol (THIPgaboxadol)

Extrasynaptic GABAA Receptorsamp drugs

Studies have begun to identify extrasynaptic GABAARs as novel targets for a diverse array of endogenous and

clinically relevant agents including

bull Certain Neuroactive steroidsbull Amino acid Taurinebull Ethanolbull Several Anestheticbull Hypnotic agentsbull Analgesicsbull Some Anticonvulsant drugs

SUBUNIT AND PHARMACOLOGICAL ACTION

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Subunits at GABA ndash A

SUBUNITS OF GABA

GABAA receptor structure

bull It is a multimeric transmembrane receptor that consists of 5 subunits arranged around a central Clminus ion pore

bull Members of family of Cys-loop ligand-gated ion channels (loop formed by a disulfide bond betn 2 cysteine residues)

bull The receptor is usually located postsynaptically However some isoforms may be found extrasynaptically

bull Each subunit is composed of a polypeptide sequence of approximately 450ndash630 amino acids (40ndash60 kDa) with large N-terminal and smaller C-terminal extracellular domains

bull individual subunits contain four distinct transmembrane(TM) domains with the second transmembrane domain (TM2) lining the channel lumen

bull A large intracellular loop connects the TM3 and TM4 regions providing sites for phosphorylationby a range of serine threonine aspargine amp tyrosine kinases

GABAA receptor MOA

bull Binding of GABA triggers opening of the Clminus ion pore

bull This drives the membrane potential towards the reversal potential of the Clmacr ion which is about ndash65 mV in neurons inhibiting the firing of new action potentials

bull This makes it more difficult for excitatory neurotransmitters to depolarize the neuron

bull The net effect is typically inhibitory reducing the activity of the neuron

Video clip

>

Extrasynaptic GABAA Receptorsbull Synaptic GABAA receptors Underlie

ldquophasicrdquo inhibitionbull By contrast extra-synaptic receptors

are usually exposed to low but persistent

GABA concentrations leading to ldquotonicrdquo inhibition

bull Compared to their synaptic counterparts these showi Increased sensitivity to GABAii Reduced propensity to desensitizeiii More rapid deactivation phase after removal of GABA

Extrasynaptic GABAA Receptors

bull The occurrence of tonic GABAA inhibition coincides with the expression of relatively rare receptor subunits articularly the α4 α6 and δ subunits and as a general rule-of-thumb δ subunit-containing receptors are extrasynaptic

bull The subunit can also govern receptor pharmacologyndash extrasynaptic GABAARs are insensitive to benzodiazepine

agonists ndash but highly sensitive to the GABAAR ldquosuper agonistrdquo

tetrahydroisoxazolopyridineol (THIPgaboxadol)

Extrasynaptic GABAA Receptorsamp drugs

Studies have begun to identify extrasynaptic GABAARs as novel targets for a diverse array of endogenous and

clinically relevant agents including

bull Certain Neuroactive steroidsbull Amino acid Taurinebull Ethanolbull Several Anestheticbull Hypnotic agentsbull Analgesicsbull Some Anticonvulsant drugs

SUBUNIT AND PHARMACOLOGICAL ACTION

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

SUBUNITS OF GABA

GABAA receptor structure

bull It is a multimeric transmembrane receptor that consists of 5 subunits arranged around a central Clminus ion pore

bull Members of family of Cys-loop ligand-gated ion channels (loop formed by a disulfide bond betn 2 cysteine residues)

bull The receptor is usually located postsynaptically However some isoforms may be found extrasynaptically

bull Each subunit is composed of a polypeptide sequence of approximately 450ndash630 amino acids (40ndash60 kDa) with large N-terminal and smaller C-terminal extracellular domains

bull individual subunits contain four distinct transmembrane(TM) domains with the second transmembrane domain (TM2) lining the channel lumen

bull A large intracellular loop connects the TM3 and TM4 regions providing sites for phosphorylationby a range of serine threonine aspargine amp tyrosine kinases

GABAA receptor MOA

bull Binding of GABA triggers opening of the Clminus ion pore

bull This drives the membrane potential towards the reversal potential of the Clmacr ion which is about ndash65 mV in neurons inhibiting the firing of new action potentials

bull This makes it more difficult for excitatory neurotransmitters to depolarize the neuron

bull The net effect is typically inhibitory reducing the activity of the neuron

Video clip

>

Extrasynaptic GABAA Receptorsbull Synaptic GABAA receptors Underlie

ldquophasicrdquo inhibitionbull By contrast extra-synaptic receptors

are usually exposed to low but persistent

GABA concentrations leading to ldquotonicrdquo inhibition

bull Compared to their synaptic counterparts these showi Increased sensitivity to GABAii Reduced propensity to desensitizeiii More rapid deactivation phase after removal of GABA

Extrasynaptic GABAA Receptors

bull The occurrence of tonic GABAA inhibition coincides with the expression of relatively rare receptor subunits articularly the α4 α6 and δ subunits and as a general rule-of-thumb δ subunit-containing receptors are extrasynaptic

bull The subunit can also govern receptor pharmacologyndash extrasynaptic GABAARs are insensitive to benzodiazepine

agonists ndash but highly sensitive to the GABAAR ldquosuper agonistrdquo

tetrahydroisoxazolopyridineol (THIPgaboxadol)

Extrasynaptic GABAA Receptorsamp drugs

Studies have begun to identify extrasynaptic GABAARs as novel targets for a diverse array of endogenous and

clinically relevant agents including

bull Certain Neuroactive steroidsbull Amino acid Taurinebull Ethanolbull Several Anestheticbull Hypnotic agentsbull Analgesicsbull Some Anticonvulsant drugs

SUBUNIT AND PHARMACOLOGICAL ACTION

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

GABAA receptor structure

bull It is a multimeric transmembrane receptor that consists of 5 subunits arranged around a central Clminus ion pore

bull Members of family of Cys-loop ligand-gated ion channels (loop formed by a disulfide bond betn 2 cysteine residues)

bull The receptor is usually located postsynaptically However some isoforms may be found extrasynaptically

bull Each subunit is composed of a polypeptide sequence of approximately 450ndash630 amino acids (40ndash60 kDa) with large N-terminal and smaller C-terminal extracellular domains

bull individual subunits contain four distinct transmembrane(TM) domains with the second transmembrane domain (TM2) lining the channel lumen

bull A large intracellular loop connects the TM3 and TM4 regions providing sites for phosphorylationby a range of serine threonine aspargine amp tyrosine kinases

GABAA receptor MOA

bull Binding of GABA triggers opening of the Clminus ion pore

bull This drives the membrane potential towards the reversal potential of the Clmacr ion which is about ndash65 mV in neurons inhibiting the firing of new action potentials

bull This makes it more difficult for excitatory neurotransmitters to depolarize the neuron

bull The net effect is typically inhibitory reducing the activity of the neuron

Video clip

>

Extrasynaptic GABAA Receptorsbull Synaptic GABAA receptors Underlie

ldquophasicrdquo inhibitionbull By contrast extra-synaptic receptors

are usually exposed to low but persistent

GABA concentrations leading to ldquotonicrdquo inhibition

bull Compared to their synaptic counterparts these showi Increased sensitivity to GABAii Reduced propensity to desensitizeiii More rapid deactivation phase after removal of GABA

Extrasynaptic GABAA Receptors

bull The occurrence of tonic GABAA inhibition coincides with the expression of relatively rare receptor subunits articularly the α4 α6 and δ subunits and as a general rule-of-thumb δ subunit-containing receptors are extrasynaptic

bull The subunit can also govern receptor pharmacologyndash extrasynaptic GABAARs are insensitive to benzodiazepine

agonists ndash but highly sensitive to the GABAAR ldquosuper agonistrdquo

tetrahydroisoxazolopyridineol (THIPgaboxadol)

Extrasynaptic GABAA Receptorsamp drugs

Studies have begun to identify extrasynaptic GABAARs as novel targets for a diverse array of endogenous and

clinically relevant agents including

bull Certain Neuroactive steroidsbull Amino acid Taurinebull Ethanolbull Several Anestheticbull Hypnotic agentsbull Analgesicsbull Some Anticonvulsant drugs

SUBUNIT AND PHARMACOLOGICAL ACTION

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

bull Each subunit is composed of a polypeptide sequence of approximately 450ndash630 amino acids (40ndash60 kDa) with large N-terminal and smaller C-terminal extracellular domains

bull individual subunits contain four distinct transmembrane(TM) domains with the second transmembrane domain (TM2) lining the channel lumen

bull A large intracellular loop connects the TM3 and TM4 regions providing sites for phosphorylationby a range of serine threonine aspargine amp tyrosine kinases

GABAA receptor MOA

bull Binding of GABA triggers opening of the Clminus ion pore

bull This drives the membrane potential towards the reversal potential of the Clmacr ion which is about ndash65 mV in neurons inhibiting the firing of new action potentials

bull This makes it more difficult for excitatory neurotransmitters to depolarize the neuron

bull The net effect is typically inhibitory reducing the activity of the neuron

Video clip

>

Extrasynaptic GABAA Receptorsbull Synaptic GABAA receptors Underlie

ldquophasicrdquo inhibitionbull By contrast extra-synaptic receptors

are usually exposed to low but persistent

GABA concentrations leading to ldquotonicrdquo inhibition

bull Compared to their synaptic counterparts these showi Increased sensitivity to GABAii Reduced propensity to desensitizeiii More rapid deactivation phase after removal of GABA

Extrasynaptic GABAA Receptors

bull The occurrence of tonic GABAA inhibition coincides with the expression of relatively rare receptor subunits articularly the α4 α6 and δ subunits and as a general rule-of-thumb δ subunit-containing receptors are extrasynaptic

bull The subunit can also govern receptor pharmacologyndash extrasynaptic GABAARs are insensitive to benzodiazepine

agonists ndash but highly sensitive to the GABAAR ldquosuper agonistrdquo

tetrahydroisoxazolopyridineol (THIPgaboxadol)

Extrasynaptic GABAA Receptorsamp drugs

Studies have begun to identify extrasynaptic GABAARs as novel targets for a diverse array of endogenous and

clinically relevant agents including

bull Certain Neuroactive steroidsbull Amino acid Taurinebull Ethanolbull Several Anestheticbull Hypnotic agentsbull Analgesicsbull Some Anticonvulsant drugs

SUBUNIT AND PHARMACOLOGICAL ACTION

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

GABAA receptor MOA

bull Binding of GABA triggers opening of the Clminus ion pore

bull This drives the membrane potential towards the reversal potential of the Clmacr ion which is about ndash65 mV in neurons inhibiting the firing of new action potentials

bull This makes it more difficult for excitatory neurotransmitters to depolarize the neuron

bull The net effect is typically inhibitory reducing the activity of the neuron

Video clip

>

Extrasynaptic GABAA Receptorsbull Synaptic GABAA receptors Underlie

ldquophasicrdquo inhibitionbull By contrast extra-synaptic receptors

are usually exposed to low but persistent

GABA concentrations leading to ldquotonicrdquo inhibition

bull Compared to their synaptic counterparts these showi Increased sensitivity to GABAii Reduced propensity to desensitizeiii More rapid deactivation phase after removal of GABA

Extrasynaptic GABAA Receptors

bull The occurrence of tonic GABAA inhibition coincides with the expression of relatively rare receptor subunits articularly the α4 α6 and δ subunits and as a general rule-of-thumb δ subunit-containing receptors are extrasynaptic

bull The subunit can also govern receptor pharmacologyndash extrasynaptic GABAARs are insensitive to benzodiazepine

agonists ndash but highly sensitive to the GABAAR ldquosuper agonistrdquo

tetrahydroisoxazolopyridineol (THIPgaboxadol)

Extrasynaptic GABAA Receptorsamp drugs

Studies have begun to identify extrasynaptic GABAARs as novel targets for a diverse array of endogenous and

clinically relevant agents including

bull Certain Neuroactive steroidsbull Amino acid Taurinebull Ethanolbull Several Anestheticbull Hypnotic agentsbull Analgesicsbull Some Anticonvulsant drugs

SUBUNIT AND PHARMACOLOGICAL ACTION

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Video clip

>

Extrasynaptic GABAA Receptorsbull Synaptic GABAA receptors Underlie

ldquophasicrdquo inhibitionbull By contrast extra-synaptic receptors

are usually exposed to low but persistent

GABA concentrations leading to ldquotonicrdquo inhibition

bull Compared to their synaptic counterparts these showi Increased sensitivity to GABAii Reduced propensity to desensitizeiii More rapid deactivation phase after removal of GABA

Extrasynaptic GABAA Receptors

bull The occurrence of tonic GABAA inhibition coincides with the expression of relatively rare receptor subunits articularly the α4 α6 and δ subunits and as a general rule-of-thumb δ subunit-containing receptors are extrasynaptic

bull The subunit can also govern receptor pharmacologyndash extrasynaptic GABAARs are insensitive to benzodiazepine

agonists ndash but highly sensitive to the GABAAR ldquosuper agonistrdquo

tetrahydroisoxazolopyridineol (THIPgaboxadol)

Extrasynaptic GABAA Receptorsamp drugs

Studies have begun to identify extrasynaptic GABAARs as novel targets for a diverse array of endogenous and

clinically relevant agents including

bull Certain Neuroactive steroidsbull Amino acid Taurinebull Ethanolbull Several Anestheticbull Hypnotic agentsbull Analgesicsbull Some Anticonvulsant drugs

SUBUNIT AND PHARMACOLOGICAL ACTION

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Extrasynaptic GABAA Receptorsbull Synaptic GABAA receptors Underlie

ldquophasicrdquo inhibitionbull By contrast extra-synaptic receptors

are usually exposed to low but persistent

GABA concentrations leading to ldquotonicrdquo inhibition

bull Compared to their synaptic counterparts these showi Increased sensitivity to GABAii Reduced propensity to desensitizeiii More rapid deactivation phase after removal of GABA

Extrasynaptic GABAA Receptors

bull The occurrence of tonic GABAA inhibition coincides with the expression of relatively rare receptor subunits articularly the α4 α6 and δ subunits and as a general rule-of-thumb δ subunit-containing receptors are extrasynaptic

bull The subunit can also govern receptor pharmacologyndash extrasynaptic GABAARs are insensitive to benzodiazepine

agonists ndash but highly sensitive to the GABAAR ldquosuper agonistrdquo

tetrahydroisoxazolopyridineol (THIPgaboxadol)

Extrasynaptic GABAA Receptorsamp drugs

Studies have begun to identify extrasynaptic GABAARs as novel targets for a diverse array of endogenous and

clinically relevant agents including

bull Certain Neuroactive steroidsbull Amino acid Taurinebull Ethanolbull Several Anestheticbull Hypnotic agentsbull Analgesicsbull Some Anticonvulsant drugs

SUBUNIT AND PHARMACOLOGICAL ACTION

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Extrasynaptic GABAA Receptors

bull The occurrence of tonic GABAA inhibition coincides with the expression of relatively rare receptor subunits articularly the α4 α6 and δ subunits and as a general rule-of-thumb δ subunit-containing receptors are extrasynaptic

bull The subunit can also govern receptor pharmacologyndash extrasynaptic GABAARs are insensitive to benzodiazepine

agonists ndash but highly sensitive to the GABAAR ldquosuper agonistrdquo

tetrahydroisoxazolopyridineol (THIPgaboxadol)

Extrasynaptic GABAA Receptorsamp drugs

Studies have begun to identify extrasynaptic GABAARs as novel targets for a diverse array of endogenous and

clinically relevant agents including

bull Certain Neuroactive steroidsbull Amino acid Taurinebull Ethanolbull Several Anestheticbull Hypnotic agentsbull Analgesicsbull Some Anticonvulsant drugs

SUBUNIT AND PHARMACOLOGICAL ACTION

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Extrasynaptic GABAA Receptorsamp drugs

Studies have begun to identify extrasynaptic GABAARs as novel targets for a diverse array of endogenous and

clinically relevant agents including

bull Certain Neuroactive steroidsbull Amino acid Taurinebull Ethanolbull Several Anestheticbull Hypnotic agentsbull Analgesicsbull Some Anticonvulsant drugs

SUBUNIT AND PHARMACOLOGICAL ACTION

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

SUBUNIT AND PHARMACOLOGICAL ACTION

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

GABAB - discovery

bull Baclofen was first synthesized in 1962 by a

chemist Heinrich Keberle and was shown to exert

potent muscle-relaxant and analgesic properties bull A report from Bowery et al holds an invaluable

pharmacological tool in elucidating the role of GABAB receptors in several disorders including epilepsy cognition defect

bull Bowery and Hudson described a bicuculline insensitive action of GABA in beclofen which lead to the discovery of GABA B later

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Molecular Structure of

GABA (B)Receptors

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Heterodimer composed of two similar subunits each with a seven trans-membrane α-helix (7 TM) topologyGABA (B) R1 and GABA (B) R2 Each subunit comprises a large N terminal extracellular domain followed by 7-transmembrane helicesand an intracellular C‑terminusGABA (B) R1 binds to ligand and initiates aconformational change in the receptor complexGABA (B) R2 interacts with and transmits thissignal to the intracellular G-protein trimer Two GABA (B) R1 isoforms GABA (B)R1a and GABA (B) R1b are expressed in the brain Two ldquoSushi motifsrdquo are present GABA (B) R1a and absent in GABA (B) R1b

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

GABA-B RECEPTOR

MOA

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

GABA-B RECEPTOR MOA

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

GABA-ρ subclass ( GABAC)

bull A subclass of ionotropic GABA receptors insensitive to typical allosteric modulators

bull GABAС receptors are exclusively composed of ρ (rho) subunits that are related to GABAA receptor subunits

bull Designated as the ρ subfamily of the GABAA receptors (GABAA-ρ)

bull These receptors are found in the retina spinal cord superior colliculus and pituitary

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

bull Three homologous ρ-subunits ρ1 to ρ3 have now been identified

bull There is only limited evidence that the ρ-subunits co-assemble with any of the other GABAA receptor subunits

bull The genes encoding the ρ1- and ρ2-subunits are found on chromosome 6 of man and are thus distinct from the clusters of receptor subunit genes which are found on

bull Chromosomes 4 5 15 and X with the exception ofδ which is found on chromosome 1

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

GABA ndashC MOA

bull Its is ionotropic receptor with action similar to GABA-A Receptor

bull These receptors are Cl- pores that are insensitive to both bicuculline and baclofen

bull They are designated GABAC in 1984 but An IUPHAR nomencleature the term GABAC be avoided and classifies it as bicuculline and baclofen-insensitive GABA receptors as a minor group

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

PHARMACOLOGICAL APPLICATION OF GABA-A

bull BZD RECEPTOR MODULATORSbull ALCOHOL amp GABAbull ROLE IN ANTIEPILEPTICSbull Neuroactive steroid

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

BASICSbull Agonists Bind to the main receptor site - also referred to as the

active or orthosteric site- and activate itbull Antagonists Bind to the main receptor site but do not activate it bull Positive Allosteric Modulators Bind to allosteric sites on the

receptor complex and affect it in a positive manner causing increased efficiency of the main site and thus increase in Cl- conductance

bull Negative Allosteric Modulators Bind to an allosteric site on the receptor complex and affect it in a negative manner causing decreased efficiency of the main site

bull Uncompetitive Channel Blockers Bind to or near the central pore of the receptor complex and directly block Cl- conductance

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

GABA ndash A Agonist

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Ibotenic acid and Muscimol

bull Contained in Amanita muscariapantherinagemmata (hallucinogenic mushroom) with muscarine muscazone

bull GABAA agonist + potent partial GABAC agonist

bull Muscimol is as much as 10 times more potent

bull Effects are frequently compared to a lucid dream state

bull Psychoactive dose of muscimol is around 10ndash15 mg

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

GABOXADOL

bull Extrasynaptic GABAA agonist

bull Increases deep sleep (stage 4)

bull BZDsZ drugs work on the α1 subtype of receptors for the neurotransmitter GABA -- thats akin to an onoff switch for the central nervous system

bull On the other hand gaboxadol works on another subtype called α4 -- its more of a dimmer switch that might help regulate sleep in a less disruptive way

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

GABAA Antagonist

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Various GABAA antagonistsDrug Detail

Bicuculline bullCompetitive antagonist of GABAA receptorsbullUtilized in laboratories in the in vitro study of epilepsybullRoutinely used to isolate glutamatergic (excitatory amino acid) receptor function

Gabazine bullAntagonist at GABAA receptorsbullUsed in scientific research and has no role in medicinebullPhasic (synaptic) inhibition is gabazine- sensitive tonic (extrasynaptic) inhibition is relatively gabazine- insensitive

Cicutoxin amp Oenantho-toxin

bullFound in various plants most notably water hemlock (Cicuta amp Oenanthe species)bullPotent noncompetitive GABA receptor antagonistbullNausea emesis and abdominal pain within 60 mins of ingestion Can lead to tremors seizures amp death

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Various GABAA antagonists

Drug Detail

Thujone bullFound in a number of plants such as arborvitae (Thuja)bullUsed in herbal medicine mainly for their immune-system stimulating effectsbullReported to be toxic to both brain and liver cells bullSide-effects include anxiety and sleeplessness seizures at high dose

Picrotoxin (cocculin)

bullPoisonous crystalline plant compoundbullFound in the fruit (fishberry) of climbing plant Anamirta cocculusbullNoncompetitive antagonist for GABAA receptor - Channel blockerbullCan be used to counter barbiturate poisoning

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Positive allosteric modulators

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

GABAA receptor allosteric MODULATORS

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Benzodiazepines (BDZ)

bull Benzodiazepines act at GABAA receptors by binding directly to a specific site distinct from that of GABA

bull They do not activate GABAA receptors directly but rather require GABA to express their effects ie they only modulate the effects of GABA

bull Studies of cloned GABAA receptors have shown that the coassembly of a γ subunit with α and β subunits confers benzodiazepine sensitivity to GABAA receptors

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Benzodiazepines bind across the interface between the α and γ subunits but only to receptors that contain γ2 and α1 α2 α3 or α5subunits1

BDZ BINDING SITEhellip

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

MOAhellip

Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor Their binding causes an allosteric (structural) modification of the receptor that results in an increase in GABA A receptor activity BDZs do not substitute for GABA which bind at the alpha sub-unit but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Binding Affinity at various subunits

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

CLINICAL USES OF BDZ MODULATORS

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

NON BENZODIAZEPINE GABA MODULATORS

bull commonly referred to as Z compounds bull They include-bull zolpidem (AMBIEN)bull zaleplon(SONATA)bull zopiclone (Not marketed in the US) and bull eszopiclone (LUNESTA) which is the S(+) enantiomer of

zopiclone

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

ZALEPLON

bull Pyrazolopyrimidine class of compound

bull Zaleplon preferentially binds to the benzodiazepine-binding site on GABAA receptors containing the α1receptor subunit

bull PK - Absorbed rapidly and reaches peak plasma concentrations in ~1 hour Its bioavailability is ~30 because of presystemic metabolism volume of distribution of ~14 Lkg and plasma-protein binding of ~60

bull Metabolised by aldehyde oxidase

bull Zaleplon (usually administered in 5- 10- or 20-mg doses) has been studied in clinical trials of patients with chronic or transient insomnia

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

ZOLPIDEM

Imidazopyridinebull Although the actions of zolpidem are

due to agonist effects on GABAA receptors and generally resemble those of benzodiazepines it produces weak anticonvulsant effects in experimental animals

bull During US clinical trials withdrawal effects within 48 hours of drug discontinuation occurred at an incidence of 1 or less Post-marketing reports of abuse dependence and withdrawal have been recorded

bull zolpidem is approved only for the short-term treatment of insomnia

bull Therapeutic doses (5 to 10 mg) zolpidem infrequently produces residual daytime sedation or amnesia adverse effects(egGI complaints or dizziness) is also low

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

ESZOPICLONE

bull Active S(+) enantiomer of zopiclone Eszopiclone has no structural similarity to benzodiazepines zolpidem or zaleplon

bull Eszopiclone is used for the long-term treatment of insomnia and for sleep maintenance It is prescribed to patients who have difficulty falling asleep as well as those who experience difficulty staying asleep and is available in (1- 2-or 3-mg)tablets

bull Eszopiclone received FDA approval based on six randomized placebo-controlled clinical trials that showed it has efficacy in treating transient and chronic insomnia

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Inverse agonist at BZD Receptor

bull Inverse agonists at the BZD site act as negative allosteric modulators of GABA-receptor function

bull Their interaction with BZD sites on the GABAA receptor can produce anxiety and seizures

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Sarmazenil bullPartial inverse agonist at the benzodiazepine site

bullIt is used in veterinary medicine to reverse the effects of benzodiazepine sedative drugs in order to rapidly re-awaken anaethetised animals

β Carbolines bullIn addition to their direct actions these molecules can block the effects of benzodiazepinesbullUses as convelsants

Negative allosteric modulators at benzodiazepine

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

BZD-site Antagonist

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST

bull Flumazenil (ROMAZICON generic) the only member of this class is an imidazobenzodiazepine that behaves as a specific benzodiazepine antagonist

bull Flumazenil binds with high affinity to specific sites on the GABA-A receptor where it competitively antagonizes the binding and allosteric effects of benzodiazepines and other ligands

bull Flumazenil antagonizes both the electrophysiological and behavioral effects of agonist and inverse-agonist benzodiazepines and B-carbolines

bull Recommended dose 1mg IV t12 of ~1 hourdose repeated till 5 mg

bull

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

FLUMAZENIL

bull Can be used to reverse the effect of benzodiazepine overdosage or to reverse the effect of benzodiazepines such as midazolam used for minor surgical procedures

bull It has been found to be effective in overdoses of non-benzodiazepine sleep enhancers - zolpidem and zaleplon

bull Use in hepatic encephalopathy amp alcohol intoxication have yielded mixed results

bull Used as a PET radioligand labeled with carbon-11 to visualize the distribution of GABAA receptors in brain

bull Flumazenil is not effective in single dose overdoses with either barbiturates or Tricyclic antidepressants rather it may cause seizures in patients poisoned with TCArsquoS

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Barbiturates

Barbiturates also facilitate the actions of GABA -A at multiple sites in the central nervous system butmdashin contrast to benzodiazepinesmdashthey appear to increase the duration of the GABA-gated chloride channel openings At Higher concentrations barbiturates directly increase chloride ion conductance

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Antiepileptic actions atGABAA Receptors

bull Modulate GABAA receptor activation

bull Phenobarbitalbull Clonazepam Diazepambull Topiramatebull Increase GABA biosynthesisbull Valproatebull Decrease GABA degradationbull Tiagabinebull Vigabatrin

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

GABAergic terminal

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Inhibition of uptake increases GABA action

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

VALPROIC ACID

bull An analogue of valeric acid

bull Believed to affect the function of the neurotransmitter GABA in the human brain by inhibition of GABA transaminase

bull Other mechanisms of action Blocks the voltage-gated Na+ channels Blocks T-type Ca++ channels Inhibits the enzyme histone deacetylase 1

(anti convulsant action)

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

VALPROIC ACIDIndications

bull Manic episodes associated with bipolar disorderbull As an anticonvulsant in

Absence seizures Juvenile myoclonic epilepsy

Tonic -clonic seizures Posttraumatic epilepsy

Complex partial seizures Lennox-Gastaut syndrome

bull Valproic acid is cytotoxic to many different cancer types through its action as a histone deacetylase inhibitor

bull Being tried in multiple myeloma glioma melanoma breast cancer cervical cancer and ovarian cancer

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

VIGABATRIN

bull VIGABATRIN is a ldquosuicide inhibitorrdquo of GABA transaminase

bull It blocks the conversion of GABA to succinic semialdehyderesulting in high intracellular GABA concentrations and increased synaptic GABA release

bull Primary indication for vigabatrin treatment of simple and complex partial seizures but it can also be used for generalised seizures

Other Uses ndash Drug refractory epilepsy and infantile spasm

Dose- 2 gm day Side Effects-1Behavioural

changes sedation Amnesia Weight gain

In 1 of cases visual field defects due to peipheral retinal

atrophy

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

TIAGABINbull Tiagabine (GABITRIL) is a

derivative of nipecotic acidbull Approved by the FDA as

adjunct therapy for partial seizures in adults

bull Tiagabine inhibits the GABA transporter GAT-1 and thereby reduces GABA uptake into neurons and glia

bull Paradoxically tiagabine has been associated with the occurrence of seizures in patients without epilepsy

bull Its contraindicated in absence seizure

Other side effects ndash Sedation fatigue tremors and confusionDose- 20-60 mg per day in 3 to 4 equally divided doses

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

GABAPENTIN amp PREGABALINbull Gabapentin (NEURONTIN others) and

pregabalin (LYRICA) are anti-seizure drugs that consist of a GABA molecule covalently bound to a lipophilic cyclohexane ring or isobutane respectively

bull Pregabalin increases neuronal GABA levels by producing a dose-dependent increase in glutamic acid decarboxylase activity

bull Gabapentin amp pregabalin bind to the α2δ (alpha2delta) subunit of the voltage-dependent calcium channel in the central nervous system and decrease the synaptic release of glutamate

Recent evidence suggest that they also function as GABAB receptor agonist

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

bull PK - Gabapentin and pregabalin are absorbed after oral administration It is neither metabolised nor bound to plasma proteins and are excreted unchanged mainly in the urine

bull Their half-lives approximate 6 hours

bull These compounds have no known interactions with other anti-seizure drugs

Therapeutic Uses Drug resistant partial seizures amp GTC also is being used for the treatment of migraine chronic pain and bipolar disorder FDA Approves Lyrica (Pregabalin) for Treatment of Neuropathic Pain in 2005Side effects - Somnolence dizziness ataxia and fatigue Dose ndash 200-300 mg TDS

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Role OF GABA RECEPTORS IN ANAESTHESIA

Anaesthetics primarily act by either enhancing inhibitorysignals or by blocking excitatory signals

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

GENERAL ANAESTHETICS

bull Currently there are 5 major inhalational and 5 intravenous anesthetics used to induce or maintain general anesthesia

bull InhalationalNitrous Oxide Isoflurane Sevoflurane Desflurane and Xenon

bull Intravenous Propofol Etomidate Ketamine Methohexital and Thiopental

Of these 10 general anesthetics except ketamine nitrous oxide and xenon other7 general anesthetics and sedatives like midazolam diazepam and lorazepam share a common target and mechanism of actionie they all enhance the function of GABA-A receptors

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

General anaesthetics

bull The extrasynapic α5szlig3γ2 receptor in hippocampus is probably assosciated with amnestic action of anaesthetic

bull Those receptors having δ subunit s at ventrobasal thalamic nucleus may be linked to intruding reversible loss of consciousness during anaesthesia

bull Since general anaesthetics are hydrophobic and need to access the CNS they target hydrophobic pockets within the transmembrane domains of the receptor

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Single amino acids determine iv anaesthetic activity

bull If GABA-A receptors are important targets for general anaestheticsare all receptors equally sensitive to their effects or are certain subunit combinations more sensitive than others

bull Etomidate which demonstrates selective effects for receptors containing certain subtypes of the szligsubunit For example receptors containing szlig2or szlig3 but not szlig1subunits are particularly sensitive to the modulatory effects of this anaesthetic4 Studies using a molecular techniques demonstrated that the selectivity of etomidate maps to a single amino acid located within the second transmembrane domain (TM2) of the b subunit (asparagine for szlig2 and szlig3 and serine for szlig1 subunits) Exchanging the asparagine for serine at szlig2 increased the sensitivity of etomidate

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

A binding site for volatile anaesthetics

bull The effects of volatile agents on GABA-A receptor activity are also influenced by a single amino acid (serine) is located within the TM2 region in the α subunit

bull Subsequent studies demonstrated that the volume of this amino acid correlated with the activity of volatile anaesthetics on GABA-A receptors Specifically substitution with small volume amino acids enhanced the potentiating activity of isofluranehalothane and chloroform whereas the presence of bulkier amino acids reduced anaesthetic activity

bull Use of volatile agents of differing size (isoflurane 144 A deg

halothane 110 A deg and chloroform 90 A deg ) led to the proposalthat the four amino acid residues from the extracellular end of TM1 TM2 TM3 and TM4 domains of the a subunit contribute towards an anaesthetic binding pocket for volatile agents

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

Neuroactive steroids

Synthesized in the central and peripheral nervous system especially in myelinating glial cells from cholesterol or steroidal precursors

bull These include pregnenolone (PREG) and dehydroepiandrosterone (DHEA) their sulfates and reduced metabolites

bull Neurosteroids have a wide range of potential clinical applications from sedation to treatment of epilepsy and traumatic brain injury

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

NEUROACTIVE STEROIDS

bull Allopregnanolone and tetrahydrodeoxycorticosterone have been surmised to enhance GABA-mediated Clmacr currents whereas pregnenolone sulfate amp dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABAA receptors

bull Ganaxolone an analog of the endogenous neurosteroid allopregnanolone is under investigation for the treatment of epilepsy and was effective in the treatment of partial seizures in adults and was tolerated

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

This animation shows one model for how neurosteroids may increase Clndash flux through a GABA-A receptor Binding of the neurosteroid (ALLO) allows a protein kinase C (PKC) phosphorylation site to become accessible Phosphorylation of the channel increases flux through the channel allowing more Clndash ions to flow through the channel in the presence of GABA than when the channel is activated by GABA without prior phosphorylation The top of this animated gif shows how the channel the ligands and G protein-activated PKC may be interacting at the plasma membrane The bottom of the animation shows how these interactions affect the current trace recorded using electrophysiological techniques In the current trace the size of the signal is smaller in the absence of neurosteroid compared to the size of the signal after exposure to neurosteroid and subsequent phosphorylation of the channel

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

ALPHAXOLONE

Neurosteroid general anaestheticA study 1987 found the primary mechanism for the anaesthetic action of alfaxalone to be modulation of neuronal cell membrane chloride ion transport induced by binding of alfaxalone to GABAA cell surface receptors It is licensed for use in both dogs and catsUnlike some of its predecessors alfaxalone is not associated with histamine release and anaphylaxis

bull A 1994 study found that alfaxalone binds to a different region of this receptor than the benzodiazepines

bull Alfaxalone is metabolised rapidly in the liver Alfaxalone has a very short plasma elimination half-life in dogs and cats

OTHER NEUROACTIVE STEROIDSDrug Details

Hydroxydione bullProved to be a useful anaesthetic drug with a good safety profilebullBut was painful and irritating when injected probably due to poor solubility

Althesin (alphaxolone + alphadolone)

bullWithdrawn from human use due to rare but serious toxic reactions but is still used in veterinary medicine

Minaxolone bullAround three times more potent than althesin without the toxicity problemsbullWithdrawn because animal studies suggested potential carcinogenicity

ALCOHOL amp GABA RECPTOR

Alcohol bull Ingestion of ethanol results in a

dose-dependent reduction of central nervous system (CNS) activity

bull For several decades it was presumed that this CNS depression was due to a major effect of ethanol is through BZD receptors on GABA

bull But a lack of ethanolrsquos effect on GABA responsiveness from isolated neurons with this receptor subtype discontinued this contention

bull Direct action of ethanol on α4β3δ or α6β3δ selectivity for separate GABA mimetic action is proved today

NEUROSTEROID INVOLVEMENT IN THEGABAMIMETIC PROFILE OF ETHANOL

Following ethanol consumption there is activation of the hypothalamicpituitary-adrenal (HPA) axis (denoted by the darkened arrows) ethanol increases neurosteroid precursors from the adrenal which in turn results inincreased neurosteroids in brain Since neuroactive steroids enhance GABA responsiveness (Lambert et al 2001 2003 Paul and Purdy 1992) it is proposed that the ethanol-induced enhancement of neurosteroid presence in brain synergizes theeffect of GABA released by ethanol (Criswell and Breese 2005)

GABA-

GLUTAMATE+

Alcohol

GABA-

GLUTAMATE+

Chronic Alcohol

ROLE OF GABA IN ALCOHOL WITHDRAWAL

Thaugh benzodiazepines have been the primary treatment modality for alcohol withdrawal

A growing number of preclinical and clinical studies suggest that the anticonvulsants like gabapentin m

Two RCT ndash First (n=60) and second (n=21) proved that gabapentin was significantly more effective than placebo for prevention of alcohol withdrawal symptoms

But except insomnia

Other drugs which are currently investigated for alcohol withdrawal effect are ndashBaclofen and GHB

GHB (Gamma hydroxy butyric acid)

Gamma-hydroxybutyric acid (GHB) gammaaminobutyricacid (GABA) that has been used in research and clinical medicine formany years

is a naturally occurring analog of

GHB (Gamma hydroxy butyric acid)

bull GHB usually exists either as a free acid or as a sodium salt The sodium salt is called Sodium Oxybate

bull and is soluble in water and methanolbull GHB as is used to treat cataplexy and excessive daytime sleepiness in

patients with narcolepsy bull MOAbull GHB has at least two distinct binding sites in the CNS

ndash Agonist at the newly-characterized GHB receptor which is excitatorybull Weak agonist at the GABAB receptor which is inhibitory Previously used

as an anesthetic in 1960s butbull was withdrawn due to its side effects like seizures andbull coma Due to its euphoric effects it is nowadays used asbull lsquoPOTENNTIAL ABUSIVE DRUGrsquo

EPIDEMIOLOGY OF GHB ABUSE

Effectshellip

NEGATIVE POINTS POSITIVE POINTSRave drug Can be used in narcolepsyDate rape drug Tried for alcohol withdrawal

Body builders addiction

Excellent dilator of cervix

Euphoria amp crime Can be used as an aphrodesiac

GHB Rage or reprive

GABAB Agonist

BACLOFEN

Derivative of GABA (β-parachlorophenyl GABA)It is primarily used to treat spasticity In disorders like multiple sclerosisALSSpinal injuries

However its relatively ineffective in stroke parkinsonismCerebral palsy and rheumatic muscle spasm

Tolerance does not develop - baclofen retains its therapeutic anti-spasmodic effects even after years of use

Baclofenbull Primary site is possibly the spinal cord

where it depresses both polysynaptic and monosynaptic reflexes

bull Also used in tardive dyskinesia and alcohol withdrawal

bull ADR - Sedation (less than BZDs) confusion weakness

bull Baclofen can be delivered directly into the space around the spinal cord by use of a surgically implanted pump and an intrathecal catheter

Excreted unchanged in urine with t12- 3 to 4 hrsDose -10 to 25 mg TDS

Phenibut

bull β-Phenyl-γ-aminobutyric acid is a derivative of GABAbull Binds the GABAB metabotropic receptor

bull The addition of a phenyl group in the β position allows phenibut to cross the blood brain barrier

bull Only the R enantiomer is biologically active

bull Sold as a dietary supplement in the United States while in Russia it is sold as a neuropsychotropic drug

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

ALCOHOL amp GABA RECPTOR

Alcohol bull Ingestion of ethanol results in a

dose-dependent reduction of central nervous system (CNS) activity

bull For several decades it was presumed that this CNS depression was due to a major effect of ethanol is through BZD receptors on GABA

bull But a lack of ethanolrsquos effect on GABA responsiveness from isolated neurons with this receptor subtype discontinued this contention

bull Direct action of ethanol on α4β3δ or α6β3δ selectivity for separate GABA mimetic action is proved today

NEUROSTEROID INVOLVEMENT IN THEGABAMIMETIC PROFILE OF ETHANOL

Following ethanol consumption there is activation of the hypothalamicpituitary-adrenal (HPA) axis (denoted by the darkened arrows) ethanol increases neurosteroid precursors from the adrenal which in turn results inincreased neurosteroids in brain Since neuroactive steroids enhance GABA responsiveness (Lambert et al 2001 2003 Paul and Purdy 1992) it is proposed that the ethanol-induced enhancement of neurosteroid presence in brain synergizes theeffect of GABA released by ethanol (Criswell and Breese 2005)

GABA-

GLUTAMATE+

Alcohol

GABA-

GLUTAMATE+

Chronic Alcohol

ROLE OF GABA IN ALCOHOL WITHDRAWAL

Thaugh benzodiazepines have been the primary treatment modality for alcohol withdrawal

A growing number of preclinical and clinical studies suggest that the anticonvulsants like gabapentin m

Two RCT ndash First (n=60) and second (n=21) proved that gabapentin was significantly more effective than placebo for prevention of alcohol withdrawal symptoms

But except insomnia

Other drugs which are currently investigated for alcohol withdrawal effect are ndashBaclofen and GHB

GHB (Gamma hydroxy butyric acid)

Gamma-hydroxybutyric acid (GHB) gammaaminobutyricacid (GABA) that has been used in research and clinical medicine formany years

is a naturally occurring analog of

GHB (Gamma hydroxy butyric acid)

bull GHB usually exists either as a free acid or as a sodium salt The sodium salt is called Sodium Oxybate

bull and is soluble in water and methanolbull GHB as is used to treat cataplexy and excessive daytime sleepiness in

patients with narcolepsy bull MOAbull GHB has at least two distinct binding sites in the CNS

ndash Agonist at the newly-characterized GHB receptor which is excitatorybull Weak agonist at the GABAB receptor which is inhibitory Previously used

as an anesthetic in 1960s butbull was withdrawn due to its side effects like seizures andbull coma Due to its euphoric effects it is nowadays used asbull lsquoPOTENNTIAL ABUSIVE DRUGrsquo

EPIDEMIOLOGY OF GHB ABUSE

Effectshellip

NEGATIVE POINTS POSITIVE POINTSRave drug Can be used in narcolepsyDate rape drug Tried for alcohol withdrawal

Body builders addiction

Excellent dilator of cervix

Euphoria amp crime Can be used as an aphrodesiac

GHB Rage or reprive

GABAB Agonist

BACLOFEN

Derivative of GABA (β-parachlorophenyl GABA)It is primarily used to treat spasticity In disorders like multiple sclerosisALSSpinal injuries

However its relatively ineffective in stroke parkinsonismCerebral palsy and rheumatic muscle spasm

Tolerance does not develop - baclofen retains its therapeutic anti-spasmodic effects even after years of use

Baclofenbull Primary site is possibly the spinal cord

where it depresses both polysynaptic and monosynaptic reflexes

bull Also used in tardive dyskinesia and alcohol withdrawal

bull ADR - Sedation (less than BZDs) confusion weakness

bull Baclofen can be delivered directly into the space around the spinal cord by use of a surgically implanted pump and an intrathecal catheter

Excreted unchanged in urine with t12- 3 to 4 hrsDose -10 to 25 mg TDS

Phenibut

bull β-Phenyl-γ-aminobutyric acid is a derivative of GABAbull Binds the GABAB metabotropic receptor

bull The addition of a phenyl group in the β position allows phenibut to cross the blood brain barrier

bull Only the R enantiomer is biologically active

bull Sold as a dietary supplement in the United States while in Russia it is sold as a neuropsychotropic drug

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Alcohol bull Ingestion of ethanol results in a

dose-dependent reduction of central nervous system (CNS) activity

bull For several decades it was presumed that this CNS depression was due to a major effect of ethanol is through BZD receptors on GABA

bull But a lack of ethanolrsquos effect on GABA responsiveness from isolated neurons with this receptor subtype discontinued this contention

bull Direct action of ethanol on α4β3δ or α6β3δ selectivity for separate GABA mimetic action is proved today

NEUROSTEROID INVOLVEMENT IN THEGABAMIMETIC PROFILE OF ETHANOL

Following ethanol consumption there is activation of the hypothalamicpituitary-adrenal (HPA) axis (denoted by the darkened arrows) ethanol increases neurosteroid precursors from the adrenal which in turn results inincreased neurosteroids in brain Since neuroactive steroids enhance GABA responsiveness (Lambert et al 2001 2003 Paul and Purdy 1992) it is proposed that the ethanol-induced enhancement of neurosteroid presence in brain synergizes theeffect of GABA released by ethanol (Criswell and Breese 2005)

GABA-

GLUTAMATE+

Alcohol

GABA-

GLUTAMATE+

Chronic Alcohol

ROLE OF GABA IN ALCOHOL WITHDRAWAL

Thaugh benzodiazepines have been the primary treatment modality for alcohol withdrawal

A growing number of preclinical and clinical studies suggest that the anticonvulsants like gabapentin m

Two RCT ndash First (n=60) and second (n=21) proved that gabapentin was significantly more effective than placebo for prevention of alcohol withdrawal symptoms

But except insomnia

Other drugs which are currently investigated for alcohol withdrawal effect are ndashBaclofen and GHB

GHB (Gamma hydroxy butyric acid)

Gamma-hydroxybutyric acid (GHB) gammaaminobutyricacid (GABA) that has been used in research and clinical medicine formany years

is a naturally occurring analog of

GHB (Gamma hydroxy butyric acid)

bull GHB usually exists either as a free acid or as a sodium salt The sodium salt is called Sodium Oxybate

bull and is soluble in water and methanolbull GHB as is used to treat cataplexy and excessive daytime sleepiness in

patients with narcolepsy bull MOAbull GHB has at least two distinct binding sites in the CNS

ndash Agonist at the newly-characterized GHB receptor which is excitatorybull Weak agonist at the GABAB receptor which is inhibitory Previously used

as an anesthetic in 1960s butbull was withdrawn due to its side effects like seizures andbull coma Due to its euphoric effects it is nowadays used asbull lsquoPOTENNTIAL ABUSIVE DRUGrsquo

EPIDEMIOLOGY OF GHB ABUSE

Effectshellip

NEGATIVE POINTS POSITIVE POINTSRave drug Can be used in narcolepsyDate rape drug Tried for alcohol withdrawal

Body builders addiction

Excellent dilator of cervix

Euphoria amp crime Can be used as an aphrodesiac

GHB Rage or reprive

GABAB Agonist

BACLOFEN

Derivative of GABA (β-parachlorophenyl GABA)It is primarily used to treat spasticity In disorders like multiple sclerosisALSSpinal injuries

However its relatively ineffective in stroke parkinsonismCerebral palsy and rheumatic muscle spasm

Tolerance does not develop - baclofen retains its therapeutic anti-spasmodic effects even after years of use

Baclofenbull Primary site is possibly the spinal cord

where it depresses both polysynaptic and monosynaptic reflexes

bull Also used in tardive dyskinesia and alcohol withdrawal

bull ADR - Sedation (less than BZDs) confusion weakness

bull Baclofen can be delivered directly into the space around the spinal cord by use of a surgically implanted pump and an intrathecal catheter

Excreted unchanged in urine with t12- 3 to 4 hrsDose -10 to 25 mg TDS

Phenibut

bull β-Phenyl-γ-aminobutyric acid is a derivative of GABAbull Binds the GABAB metabotropic receptor

bull The addition of a phenyl group in the β position allows phenibut to cross the blood brain barrier

bull Only the R enantiomer is biologically active

bull Sold as a dietary supplement in the United States while in Russia it is sold as a neuropsychotropic drug

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

NEUROSTEROID INVOLVEMENT IN THEGABAMIMETIC PROFILE OF ETHANOL

Following ethanol consumption there is activation of the hypothalamicpituitary-adrenal (HPA) axis (denoted by the darkened arrows) ethanol increases neurosteroid precursors from the adrenal which in turn results inincreased neurosteroids in brain Since neuroactive steroids enhance GABA responsiveness (Lambert et al 2001 2003 Paul and Purdy 1992) it is proposed that the ethanol-induced enhancement of neurosteroid presence in brain synergizes theeffect of GABA released by ethanol (Criswell and Breese 2005)

GABA-

GLUTAMATE+

Alcohol

GABA-

GLUTAMATE+

Chronic Alcohol

ROLE OF GABA IN ALCOHOL WITHDRAWAL

Thaugh benzodiazepines have been the primary treatment modality for alcohol withdrawal

A growing number of preclinical and clinical studies suggest that the anticonvulsants like gabapentin m

Two RCT ndash First (n=60) and second (n=21) proved that gabapentin was significantly more effective than placebo for prevention of alcohol withdrawal symptoms

But except insomnia

Other drugs which are currently investigated for alcohol withdrawal effect are ndashBaclofen and GHB

GHB (Gamma hydroxy butyric acid)

Gamma-hydroxybutyric acid (GHB) gammaaminobutyricacid (GABA) that has been used in research and clinical medicine formany years

is a naturally occurring analog of

GHB (Gamma hydroxy butyric acid)

bull GHB usually exists either as a free acid or as a sodium salt The sodium salt is called Sodium Oxybate

bull and is soluble in water and methanolbull GHB as is used to treat cataplexy and excessive daytime sleepiness in

patients with narcolepsy bull MOAbull GHB has at least two distinct binding sites in the CNS

ndash Agonist at the newly-characterized GHB receptor which is excitatorybull Weak agonist at the GABAB receptor which is inhibitory Previously used

as an anesthetic in 1960s butbull was withdrawn due to its side effects like seizures andbull coma Due to its euphoric effects it is nowadays used asbull lsquoPOTENNTIAL ABUSIVE DRUGrsquo

EPIDEMIOLOGY OF GHB ABUSE

Effectshellip

NEGATIVE POINTS POSITIVE POINTSRave drug Can be used in narcolepsyDate rape drug Tried for alcohol withdrawal

Body builders addiction

Excellent dilator of cervix

Euphoria amp crime Can be used as an aphrodesiac

GHB Rage or reprive

GABAB Agonist

BACLOFEN

Derivative of GABA (β-parachlorophenyl GABA)It is primarily used to treat spasticity In disorders like multiple sclerosisALSSpinal injuries

However its relatively ineffective in stroke parkinsonismCerebral palsy and rheumatic muscle spasm

Tolerance does not develop - baclofen retains its therapeutic anti-spasmodic effects even after years of use

Baclofenbull Primary site is possibly the spinal cord

where it depresses both polysynaptic and monosynaptic reflexes

bull Also used in tardive dyskinesia and alcohol withdrawal

bull ADR - Sedation (less than BZDs) confusion weakness

bull Baclofen can be delivered directly into the space around the spinal cord by use of a surgically implanted pump and an intrathecal catheter

Excreted unchanged in urine with t12- 3 to 4 hrsDose -10 to 25 mg TDS

Phenibut

bull β-Phenyl-γ-aminobutyric acid is a derivative of GABAbull Binds the GABAB metabotropic receptor

bull The addition of a phenyl group in the β position allows phenibut to cross the blood brain barrier

bull Only the R enantiomer is biologically active

bull Sold as a dietary supplement in the United States while in Russia it is sold as a neuropsychotropic drug

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

GABA-

GLUTAMATE+

Alcohol

GABA-

GLUTAMATE+

Chronic Alcohol

ROLE OF GABA IN ALCOHOL WITHDRAWAL

Thaugh benzodiazepines have been the primary treatment modality for alcohol withdrawal

A growing number of preclinical and clinical studies suggest that the anticonvulsants like gabapentin m

Two RCT ndash First (n=60) and second (n=21) proved that gabapentin was significantly more effective than placebo for prevention of alcohol withdrawal symptoms

But except insomnia

Other drugs which are currently investigated for alcohol withdrawal effect are ndashBaclofen and GHB

GHB (Gamma hydroxy butyric acid)

Gamma-hydroxybutyric acid (GHB) gammaaminobutyricacid (GABA) that has been used in research and clinical medicine formany years

is a naturally occurring analog of

GHB (Gamma hydroxy butyric acid)

bull GHB usually exists either as a free acid or as a sodium salt The sodium salt is called Sodium Oxybate

bull and is soluble in water and methanolbull GHB as is used to treat cataplexy and excessive daytime sleepiness in

patients with narcolepsy bull MOAbull GHB has at least two distinct binding sites in the CNS

ndash Agonist at the newly-characterized GHB receptor which is excitatorybull Weak agonist at the GABAB receptor which is inhibitory Previously used

as an anesthetic in 1960s butbull was withdrawn due to its side effects like seizures andbull coma Due to its euphoric effects it is nowadays used asbull lsquoPOTENNTIAL ABUSIVE DRUGrsquo

EPIDEMIOLOGY OF GHB ABUSE

Effectshellip

NEGATIVE POINTS POSITIVE POINTSRave drug Can be used in narcolepsyDate rape drug Tried for alcohol withdrawal

Body builders addiction

Excellent dilator of cervix

Euphoria amp crime Can be used as an aphrodesiac

GHB Rage or reprive

GABAB Agonist

BACLOFEN

Derivative of GABA (β-parachlorophenyl GABA)It is primarily used to treat spasticity In disorders like multiple sclerosisALSSpinal injuries

However its relatively ineffective in stroke parkinsonismCerebral palsy and rheumatic muscle spasm

Tolerance does not develop - baclofen retains its therapeutic anti-spasmodic effects even after years of use

Baclofenbull Primary site is possibly the spinal cord

where it depresses both polysynaptic and monosynaptic reflexes

bull Also used in tardive dyskinesia and alcohol withdrawal

bull ADR - Sedation (less than BZDs) confusion weakness

bull Baclofen can be delivered directly into the space around the spinal cord by use of a surgically implanted pump and an intrathecal catheter

Excreted unchanged in urine with t12- 3 to 4 hrsDose -10 to 25 mg TDS

Phenibut

bull β-Phenyl-γ-aminobutyric acid is a derivative of GABAbull Binds the GABAB metabotropic receptor

bull The addition of a phenyl group in the β position allows phenibut to cross the blood brain barrier

bull Only the R enantiomer is biologically active

bull Sold as a dietary supplement in the United States while in Russia it is sold as a neuropsychotropic drug

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

GABA-

GLUTAMATE+

Chronic Alcohol

ROLE OF GABA IN ALCOHOL WITHDRAWAL

Thaugh benzodiazepines have been the primary treatment modality for alcohol withdrawal

A growing number of preclinical and clinical studies suggest that the anticonvulsants like gabapentin m

Two RCT ndash First (n=60) and second (n=21) proved that gabapentin was significantly more effective than placebo for prevention of alcohol withdrawal symptoms

But except insomnia

Other drugs which are currently investigated for alcohol withdrawal effect are ndashBaclofen and GHB

GHB (Gamma hydroxy butyric acid)

Gamma-hydroxybutyric acid (GHB) gammaaminobutyricacid (GABA) that has been used in research and clinical medicine formany years

is a naturally occurring analog of

GHB (Gamma hydroxy butyric acid)

bull GHB usually exists either as a free acid or as a sodium salt The sodium salt is called Sodium Oxybate

bull and is soluble in water and methanolbull GHB as is used to treat cataplexy and excessive daytime sleepiness in

patients with narcolepsy bull MOAbull GHB has at least two distinct binding sites in the CNS

ndash Agonist at the newly-characterized GHB receptor which is excitatorybull Weak agonist at the GABAB receptor which is inhibitory Previously used

as an anesthetic in 1960s butbull was withdrawn due to its side effects like seizures andbull coma Due to its euphoric effects it is nowadays used asbull lsquoPOTENNTIAL ABUSIVE DRUGrsquo

EPIDEMIOLOGY OF GHB ABUSE

Effectshellip

NEGATIVE POINTS POSITIVE POINTSRave drug Can be used in narcolepsyDate rape drug Tried for alcohol withdrawal

Body builders addiction

Excellent dilator of cervix

Euphoria amp crime Can be used as an aphrodesiac

GHB Rage or reprive

GABAB Agonist

BACLOFEN

Derivative of GABA (β-parachlorophenyl GABA)It is primarily used to treat spasticity In disorders like multiple sclerosisALSSpinal injuries

However its relatively ineffective in stroke parkinsonismCerebral palsy and rheumatic muscle spasm

Tolerance does not develop - baclofen retains its therapeutic anti-spasmodic effects even after years of use

Baclofenbull Primary site is possibly the spinal cord

where it depresses both polysynaptic and monosynaptic reflexes

bull Also used in tardive dyskinesia and alcohol withdrawal

bull ADR - Sedation (less than BZDs) confusion weakness

bull Baclofen can be delivered directly into the space around the spinal cord by use of a surgically implanted pump and an intrathecal catheter

Excreted unchanged in urine with t12- 3 to 4 hrsDose -10 to 25 mg TDS

Phenibut

bull β-Phenyl-γ-aminobutyric acid is a derivative of GABAbull Binds the GABAB metabotropic receptor

bull The addition of a phenyl group in the β position allows phenibut to cross the blood brain barrier

bull Only the R enantiomer is biologically active

bull Sold as a dietary supplement in the United States while in Russia it is sold as a neuropsychotropic drug

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

ROLE OF GABA IN ALCOHOL WITHDRAWAL

Thaugh benzodiazepines have been the primary treatment modality for alcohol withdrawal

A growing number of preclinical and clinical studies suggest that the anticonvulsants like gabapentin m

Two RCT ndash First (n=60) and second (n=21) proved that gabapentin was significantly more effective than placebo for prevention of alcohol withdrawal symptoms

But except insomnia

Other drugs which are currently investigated for alcohol withdrawal effect are ndashBaclofen and GHB

GHB (Gamma hydroxy butyric acid)

Gamma-hydroxybutyric acid (GHB) gammaaminobutyricacid (GABA) that has been used in research and clinical medicine formany years

is a naturally occurring analog of

GHB (Gamma hydroxy butyric acid)

bull GHB usually exists either as a free acid or as a sodium salt The sodium salt is called Sodium Oxybate

bull and is soluble in water and methanolbull GHB as is used to treat cataplexy and excessive daytime sleepiness in

patients with narcolepsy bull MOAbull GHB has at least two distinct binding sites in the CNS

ndash Agonist at the newly-characterized GHB receptor which is excitatorybull Weak agonist at the GABAB receptor which is inhibitory Previously used

as an anesthetic in 1960s butbull was withdrawn due to its side effects like seizures andbull coma Due to its euphoric effects it is nowadays used asbull lsquoPOTENNTIAL ABUSIVE DRUGrsquo

EPIDEMIOLOGY OF GHB ABUSE

Effectshellip

NEGATIVE POINTS POSITIVE POINTSRave drug Can be used in narcolepsyDate rape drug Tried for alcohol withdrawal

Body builders addiction

Excellent dilator of cervix

Euphoria amp crime Can be used as an aphrodesiac

GHB Rage or reprive

GABAB Agonist

BACLOFEN

Derivative of GABA (β-parachlorophenyl GABA)It is primarily used to treat spasticity In disorders like multiple sclerosisALSSpinal injuries

However its relatively ineffective in stroke parkinsonismCerebral palsy and rheumatic muscle spasm

Tolerance does not develop - baclofen retains its therapeutic anti-spasmodic effects even after years of use

Baclofenbull Primary site is possibly the spinal cord

where it depresses both polysynaptic and monosynaptic reflexes

bull Also used in tardive dyskinesia and alcohol withdrawal

bull ADR - Sedation (less than BZDs) confusion weakness

bull Baclofen can be delivered directly into the space around the spinal cord by use of a surgically implanted pump and an intrathecal catheter

Excreted unchanged in urine with t12- 3 to 4 hrsDose -10 to 25 mg TDS

Phenibut

bull β-Phenyl-γ-aminobutyric acid is a derivative of GABAbull Binds the GABAB metabotropic receptor

bull The addition of a phenyl group in the β position allows phenibut to cross the blood brain barrier

bull Only the R enantiomer is biologically active

bull Sold as a dietary supplement in the United States while in Russia it is sold as a neuropsychotropic drug

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

GHB (Gamma hydroxy butyric acid)

Gamma-hydroxybutyric acid (GHB) gammaaminobutyricacid (GABA) that has been used in research and clinical medicine formany years

is a naturally occurring analog of

GHB (Gamma hydroxy butyric acid)

bull GHB usually exists either as a free acid or as a sodium salt The sodium salt is called Sodium Oxybate

bull and is soluble in water and methanolbull GHB as is used to treat cataplexy and excessive daytime sleepiness in

patients with narcolepsy bull MOAbull GHB has at least two distinct binding sites in the CNS

ndash Agonist at the newly-characterized GHB receptor which is excitatorybull Weak agonist at the GABAB receptor which is inhibitory Previously used

as an anesthetic in 1960s butbull was withdrawn due to its side effects like seizures andbull coma Due to its euphoric effects it is nowadays used asbull lsquoPOTENNTIAL ABUSIVE DRUGrsquo

EPIDEMIOLOGY OF GHB ABUSE

Effectshellip

NEGATIVE POINTS POSITIVE POINTSRave drug Can be used in narcolepsyDate rape drug Tried for alcohol withdrawal

Body builders addiction

Excellent dilator of cervix

Euphoria amp crime Can be used as an aphrodesiac

GHB Rage or reprive

GABAB Agonist

BACLOFEN

Derivative of GABA (β-parachlorophenyl GABA)It is primarily used to treat spasticity In disorders like multiple sclerosisALSSpinal injuries

However its relatively ineffective in stroke parkinsonismCerebral palsy and rheumatic muscle spasm

Tolerance does not develop - baclofen retains its therapeutic anti-spasmodic effects even after years of use

Baclofenbull Primary site is possibly the spinal cord

where it depresses both polysynaptic and monosynaptic reflexes

bull Also used in tardive dyskinesia and alcohol withdrawal

bull ADR - Sedation (less than BZDs) confusion weakness

bull Baclofen can be delivered directly into the space around the spinal cord by use of a surgically implanted pump and an intrathecal catheter

Excreted unchanged in urine with t12- 3 to 4 hrsDose -10 to 25 mg TDS

Phenibut

bull β-Phenyl-γ-aminobutyric acid is a derivative of GABAbull Binds the GABAB metabotropic receptor

bull The addition of a phenyl group in the β position allows phenibut to cross the blood brain barrier

bull Only the R enantiomer is biologically active

bull Sold as a dietary supplement in the United States while in Russia it is sold as a neuropsychotropic drug

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

GHB (Gamma hydroxy butyric acid)

bull GHB usually exists either as a free acid or as a sodium salt The sodium salt is called Sodium Oxybate

bull and is soluble in water and methanolbull GHB as is used to treat cataplexy and excessive daytime sleepiness in

patients with narcolepsy bull MOAbull GHB has at least two distinct binding sites in the CNS

ndash Agonist at the newly-characterized GHB receptor which is excitatorybull Weak agonist at the GABAB receptor which is inhibitory Previously used

as an anesthetic in 1960s butbull was withdrawn due to its side effects like seizures andbull coma Due to its euphoric effects it is nowadays used asbull lsquoPOTENNTIAL ABUSIVE DRUGrsquo

EPIDEMIOLOGY OF GHB ABUSE

Effectshellip

NEGATIVE POINTS POSITIVE POINTSRave drug Can be used in narcolepsyDate rape drug Tried for alcohol withdrawal

Body builders addiction

Excellent dilator of cervix

Euphoria amp crime Can be used as an aphrodesiac

GHB Rage or reprive

GABAB Agonist

BACLOFEN

Derivative of GABA (β-parachlorophenyl GABA)It is primarily used to treat spasticity In disorders like multiple sclerosisALSSpinal injuries

However its relatively ineffective in stroke parkinsonismCerebral palsy and rheumatic muscle spasm

Tolerance does not develop - baclofen retains its therapeutic anti-spasmodic effects even after years of use

Baclofenbull Primary site is possibly the spinal cord

where it depresses both polysynaptic and monosynaptic reflexes

bull Also used in tardive dyskinesia and alcohol withdrawal

bull ADR - Sedation (less than BZDs) confusion weakness

bull Baclofen can be delivered directly into the space around the spinal cord by use of a surgically implanted pump and an intrathecal catheter

Excreted unchanged in urine with t12- 3 to 4 hrsDose -10 to 25 mg TDS

Phenibut

bull β-Phenyl-γ-aminobutyric acid is a derivative of GABAbull Binds the GABAB metabotropic receptor

bull The addition of a phenyl group in the β position allows phenibut to cross the blood brain barrier

bull Only the R enantiomer is biologically active

bull Sold as a dietary supplement in the United States while in Russia it is sold as a neuropsychotropic drug

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

EPIDEMIOLOGY OF GHB ABUSE

Effectshellip

NEGATIVE POINTS POSITIVE POINTSRave drug Can be used in narcolepsyDate rape drug Tried for alcohol withdrawal

Body builders addiction

Excellent dilator of cervix

Euphoria amp crime Can be used as an aphrodesiac

GHB Rage or reprive

GABAB Agonist

BACLOFEN

Derivative of GABA (β-parachlorophenyl GABA)It is primarily used to treat spasticity In disorders like multiple sclerosisALSSpinal injuries

However its relatively ineffective in stroke parkinsonismCerebral palsy and rheumatic muscle spasm

Tolerance does not develop - baclofen retains its therapeutic anti-spasmodic effects even after years of use

Baclofenbull Primary site is possibly the spinal cord

where it depresses both polysynaptic and monosynaptic reflexes

bull Also used in tardive dyskinesia and alcohol withdrawal

bull ADR - Sedation (less than BZDs) confusion weakness

bull Baclofen can be delivered directly into the space around the spinal cord by use of a surgically implanted pump and an intrathecal catheter

Excreted unchanged in urine with t12- 3 to 4 hrsDose -10 to 25 mg TDS

Phenibut

bull β-Phenyl-γ-aminobutyric acid is a derivative of GABAbull Binds the GABAB metabotropic receptor

bull The addition of a phenyl group in the β position allows phenibut to cross the blood brain barrier

bull Only the R enantiomer is biologically active

bull Sold as a dietary supplement in the United States while in Russia it is sold as a neuropsychotropic drug

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Effectshellip

NEGATIVE POINTS POSITIVE POINTSRave drug Can be used in narcolepsyDate rape drug Tried for alcohol withdrawal

Body builders addiction

Excellent dilator of cervix

Euphoria amp crime Can be used as an aphrodesiac

GHB Rage or reprive

GABAB Agonist

BACLOFEN

Derivative of GABA (β-parachlorophenyl GABA)It is primarily used to treat spasticity In disorders like multiple sclerosisALSSpinal injuries

However its relatively ineffective in stroke parkinsonismCerebral palsy and rheumatic muscle spasm

Tolerance does not develop - baclofen retains its therapeutic anti-spasmodic effects even after years of use

Baclofenbull Primary site is possibly the spinal cord

where it depresses both polysynaptic and monosynaptic reflexes

bull Also used in tardive dyskinesia and alcohol withdrawal

bull ADR - Sedation (less than BZDs) confusion weakness

bull Baclofen can be delivered directly into the space around the spinal cord by use of a surgically implanted pump and an intrathecal catheter

Excreted unchanged in urine with t12- 3 to 4 hrsDose -10 to 25 mg TDS

Phenibut

bull β-Phenyl-γ-aminobutyric acid is a derivative of GABAbull Binds the GABAB metabotropic receptor

bull The addition of a phenyl group in the β position allows phenibut to cross the blood brain barrier

bull Only the R enantiomer is biologically active

bull Sold as a dietary supplement in the United States while in Russia it is sold as a neuropsychotropic drug

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

NEGATIVE POINTS POSITIVE POINTSRave drug Can be used in narcolepsyDate rape drug Tried for alcohol withdrawal

Body builders addiction

Excellent dilator of cervix

Euphoria amp crime Can be used as an aphrodesiac

GHB Rage or reprive

GABAB Agonist

BACLOFEN

Derivative of GABA (β-parachlorophenyl GABA)It is primarily used to treat spasticity In disorders like multiple sclerosisALSSpinal injuries

However its relatively ineffective in stroke parkinsonismCerebral palsy and rheumatic muscle spasm

Tolerance does not develop - baclofen retains its therapeutic anti-spasmodic effects even after years of use

Baclofenbull Primary site is possibly the spinal cord

where it depresses both polysynaptic and monosynaptic reflexes

bull Also used in tardive dyskinesia and alcohol withdrawal

bull ADR - Sedation (less than BZDs) confusion weakness

bull Baclofen can be delivered directly into the space around the spinal cord by use of a surgically implanted pump and an intrathecal catheter

Excreted unchanged in urine with t12- 3 to 4 hrsDose -10 to 25 mg TDS

Phenibut

bull β-Phenyl-γ-aminobutyric acid is a derivative of GABAbull Binds the GABAB metabotropic receptor

bull The addition of a phenyl group in the β position allows phenibut to cross the blood brain barrier

bull Only the R enantiomer is biologically active

bull Sold as a dietary supplement in the United States while in Russia it is sold as a neuropsychotropic drug

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

GABAB Agonist

BACLOFEN

Derivative of GABA (β-parachlorophenyl GABA)It is primarily used to treat spasticity In disorders like multiple sclerosisALSSpinal injuries

However its relatively ineffective in stroke parkinsonismCerebral palsy and rheumatic muscle spasm

Tolerance does not develop - baclofen retains its therapeutic anti-spasmodic effects even after years of use

Baclofenbull Primary site is possibly the spinal cord

where it depresses both polysynaptic and monosynaptic reflexes

bull Also used in tardive dyskinesia and alcohol withdrawal

bull ADR - Sedation (less than BZDs) confusion weakness

bull Baclofen can be delivered directly into the space around the spinal cord by use of a surgically implanted pump and an intrathecal catheter

Excreted unchanged in urine with t12- 3 to 4 hrsDose -10 to 25 mg TDS

Phenibut

bull β-Phenyl-γ-aminobutyric acid is a derivative of GABAbull Binds the GABAB metabotropic receptor

bull The addition of a phenyl group in the β position allows phenibut to cross the blood brain barrier

bull Only the R enantiomer is biologically active

bull Sold as a dietary supplement in the United States while in Russia it is sold as a neuropsychotropic drug

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

BACLOFEN

Derivative of GABA (β-parachlorophenyl GABA)It is primarily used to treat spasticity In disorders like multiple sclerosisALSSpinal injuries

However its relatively ineffective in stroke parkinsonismCerebral palsy and rheumatic muscle spasm

Tolerance does not develop - baclofen retains its therapeutic anti-spasmodic effects even after years of use

Baclofenbull Primary site is possibly the spinal cord

where it depresses both polysynaptic and monosynaptic reflexes

bull Also used in tardive dyskinesia and alcohol withdrawal

bull ADR - Sedation (less than BZDs) confusion weakness

bull Baclofen can be delivered directly into the space around the spinal cord by use of a surgically implanted pump and an intrathecal catheter

Excreted unchanged in urine with t12- 3 to 4 hrsDose -10 to 25 mg TDS

Phenibut

bull β-Phenyl-γ-aminobutyric acid is a derivative of GABAbull Binds the GABAB metabotropic receptor

bull The addition of a phenyl group in the β position allows phenibut to cross the blood brain barrier

bull Only the R enantiomer is biologically active

bull Sold as a dietary supplement in the United States while in Russia it is sold as a neuropsychotropic drug

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Baclofenbull Primary site is possibly the spinal cord

where it depresses both polysynaptic and monosynaptic reflexes

bull Also used in tardive dyskinesia and alcohol withdrawal

bull ADR - Sedation (less than BZDs) confusion weakness

bull Baclofen can be delivered directly into the space around the spinal cord by use of a surgically implanted pump and an intrathecal catheter

Excreted unchanged in urine with t12- 3 to 4 hrsDose -10 to 25 mg TDS

Phenibut

bull β-Phenyl-γ-aminobutyric acid is a derivative of GABAbull Binds the GABAB metabotropic receptor

bull The addition of a phenyl group in the β position allows phenibut to cross the blood brain barrier

bull Only the R enantiomer is biologically active

bull Sold as a dietary supplement in the United States while in Russia it is sold as a neuropsychotropic drug

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Phenibut

bull β-Phenyl-γ-aminobutyric acid is a derivative of GABAbull Binds the GABAB metabotropic receptor

bull The addition of a phenyl group in the β position allows phenibut to cross the blood brain barrier

bull Only the R enantiomer is biologically active

bull Sold as a dietary supplement in the United States while in Russia it is sold as a neuropsychotropic drug

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Phenibut

bull Structurally similar to baclofen amp phenylethylamine ndash Pharmacological effects of phenibut are similar to

baclofen but less potent ndash Additionally can function as a phenylethylamine receptor

antagonistndash Furthermore phenibut has been shown to enhance levels of

dopamine

bull Has anxiolytic effects in both animal models and in humans

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Phenibut

bull Used to treat a wide range of ailments including post-traumatic stress disorder anxiety and insomnia

bull It has been reported by some to possess neurotropic actions for its ability to improve neurological functions

bull ADR - Sedation

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

GABAB Antagonist

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Saclofen amp Phaclofen

bull These are competitive antagonist at GABAB receptor

bull This drug is an analogue of the GABAB agonist Baclofen

bull The action of saclofen on the CNS is understandably modest because G-proteins rely on an enzyme cascade

bull However in animal experiments saclofen is paradoxically observed to have an antiepileptic effect

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

GABA analogues

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Progabide

bull Analog and prodrug of GABA used in the treatment of epilepsy

bull Agonist at both the GABAA and GABAB receptors

bull Approved for either monotherapy or adjunctive use in the treatment of epilepsymdashspecifically generalized tonic-clonic myoclonic partial and Lennox-Gastaut syndrome seizuresmdashin both children and adults

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Progabide

bull Also being investigated forndash Parkinsons diseasendash Schizophreniandash Depressionndash Anxiety disorder ndash Spasticity

with various levels of success

bull Tolgabide - analogue of progabide and acts similarly to it as a prodrug of GABA and therefore as an indirect agonist of the GABA receptors

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Picamilon

bull Dietary supplement formed by combining niacin with GABAIt was developed in the Soviet Union in 1969 by the All-Union Vitamins Scientific Research Institute

bull Crosses BBB and is hydrolyzed into GABA and niacin The released GABA would activate GABA receptors potentially producing an anxiolytic response

bull The second released component niacin acts as a strong vasodilator which might be useful for the treatment of migraine headaches

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Picamilon

In Russia Picamilon is used for treatment of these illness

1 Ischemic stroke2 Asthenia3 Depression4 Senile psychosis5 Alcohol intoxication6 Migraine7 Craniocerebral trauma8 Neuroinfections9 Primary open-angle glaucoma

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

GABA ndashC AGONIST

bull CACA ndash C Amino caproic acid Weak agonistbull TACA ndash Trans isomer of CACAstrongest agonist at GABA ndash

C receptorbull R- CAMP- Recemic mixture of C-AMP acts as agonist

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

TPMPA Selective GABA-CAntagonist

bull Tetrahydropyridine ring of isoguvacine was unfavourable for interactions with GABAB receptors while the

bull methylphosphinic moiety of 3-APMPA was unfavourable for interactions with GABA-A receptors led to the synthesisof TPMPA which incorporates both the tetrahydropyridine and methylphosphinic acid moieties in the one compound

bull TPMPA has been used to provide evidence of functional GABAC receptors in the rat superior colliculus

bull mediating disinhibition in cells in the gut involved in neuroendocrine secretion and in rat spinal cord

bull TPMPA has been shown to enhance memory in chicks

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

GABA C

bull Recombinant receptor technology The cloning of ρ1 and ρ2 cDNAs from a human retinal library enabled expression of receptors in Xenopus oocytes that showed the characteristics expected of GABAC receptors

bull GABAC receptors are expected to mediate the lateral inhibition of light responses and have been shown to inhibit transmitter release at bipolar cell terminals

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

CRITERIA GABA-A GABA-B GABA-C

Distribution Mammalian CNS Cerebellum amp interpeduncular nuclei

retina spinal cordsuperior colliculus pituitary and gastrointestinaltract

CATEGORY LGCC GPCR LGCC

AGONIST GABA MuscimolTHIP

BaclofenSKF 97541

CACA GABAIsoguvacine(p)

MODULATOR BenzodiazepinesAnaestheticsBarbiturateAlcoholNeuroactive steroidshelliphellip

Zinc

ANTAGONIST BicucillinPicrotoxinGabazine

PhaclofenSaclofen

TPMPATHIPPicrotoxinLow concZinc ion

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Section III Herbal Preperations and GABA Receptors

bull Many herbal medicines are used to influence brain function in order to treat anxiety cognitive disorders and insomnia involving GABA receptor

bull Usually Herbal products act as second order modulators

iethey enhance the effect of first order modulators

bull GABA itself is an important plant constituent and

thus it should not be surprising that plants contain a

range of other chemicals that can influence GABA

function

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Bicuculline

First isolated from the plant Dicentra cucullaria and subsequently from a variety of CorydalisDicentra and Adlumia species

Competitive antagonist of GABAA receptor activation

bull Utilized in laboratories in the in vitro study of epilepsy

bull Routinely used to isolate glutamatergic (excitatory amino acid) receptor function

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Picrotoxin

Picrotoxin is an equimolar mixture of picrotoxinin and picrotin isolated from Anamirta cocculus and related poisonous plants of the moonseed family

Picrotoxinin is relatively nonspecific in that it is a potent antagonist at GABA-A and GABA-C moderate at glycine and weak at 5HT3 receptors

Can be used to counter barbiturate poisoning It is clear that bicuculline and picrotoxinin act at different sites to antagonize GABA

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Muscimolbull Muscimol is one of the most widely

used agonists in the investigation of ionotropic GABA receptors

bull It is a more potent agonist at GABA-C receptors than at GABA-A receptors

bull The agonist action of muscimol at GABA-C receptors is not blocked by bicuculline but is sensitive to picrotoxin

bull (Gaboxadol) is a conformationally restricted analogue of muscimol with analgesic and sleep-promoting properties

It was investigated for the treatment of insomnia but was recently withdrawn from phase III clinical trials due to efficacy and side-effect problems

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Apigenin ndash GABA Receptor Modulator

bull Common flavonoid found in a range of plants including chamomile tea(Matricariarecutita)

bull The chamomile tea used in treatment for insomnia and anxiety led to investigations of its active constituents including apigeninApigenin was found to have anxiolytic propertiesand it competitively inhibited the binding of flunitrazepam to brain membranes

bull Enhancement of the diazepam-induced positive allosteric modulation of GABA responses by lower concentrations of

apigenin described as a second-order modulation

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Epigallocatechin gallate (EGCG)

bull Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis)

bull Studies on α1szlig2γ2GABA-A receptors showed that EGCG at low concentrations has a potent second-order modulatory action on the first-order modulation by diazepam ( more than apegenin)

bull In well-controlled epidemiological studies it alters brain-aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders

eg Parkinsonrsquos and Alzheimerrsquos diseases

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Hispidulin and Related Flavonoids

bull Hispidulin (the 6-methoxy derivative ofbull apigenin) was isolated together with apigenin frombull Salvia officinalis (sage) recently using a benzodiazepine binding assay-

guided fractionationbull Hispidulin was some 30 times more potent than apigenin in displacing

flumazenil bindingbull Used in herbal medicine to assist memorybull An extract of Salvia lavandulaefolia (Spanish sage) has

been shown to enhance memory in healthy young volunteers

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Section IV

CURRENT KNOWEDGE SCENARIO amp FUTURE

TRENDS

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

GAD 65 OR GAD 67

Knockout of GAD65 has major impact on synaptic

GABA synthesized from astrocyte-derived

Glutamine1

bull Two isoforms GAD- 65 amp GAD- 67bull GAD65 is crucial for maintenance of biosynthesis of synaptic

GABA particularly by direct synthesis from astrocytic glutamine via glutamate

bull The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

GENE ORGANISATION OF SUBUNITS

The majority of genes coding for theGABA-A receptor subunits are organized into four clusters on chromosomes 4 5 15 and X in the human genome

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

bull Pharmacological analysis of GABA receptor was simplified after introduction of animal models in which particular GABA- A receptor subunits are either inactivated (knock out) or selectively point mutated ( knock in)

KNOCK OUT SUBUNIT

RESPONSE

α6 Motor impairing action of mice on rota rod to daiazapine

szlig3 Die in neonatal periodEpileptic seizure

γ2 Sleep time was prolongedNo action of benzodiazapines

δ Increased sleep time and convulsionsNo action of alcohol

GENE KNOCKOUT amp KNOCK IN TECHNIQUE

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

GABA OLD RECEPTOR NEW TARGETS

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

GABA ndash AS IMMUNOMODULATOR

bull In lymphocytesexposure to GABA reduced but did not abolish the transient increase in the intracellular calcium concentration that was associated with activation of the cells (Alam et al2006)

bull GABA activated GABA-A ionchannel currents in T cells and macrophages (Bjurstom et al 2008 Bhat et al 2010

bull GABA application resulted in decreased cytokine secretion and T cells proliferation (Mendu et al 2011)

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

GABA ndash AS IMMUNOMODULATOR

bull GABA appears to have a role in autoimmune diseasesbull like MS type 1 diabetes and rheumatoid arthritis and

maymodulate the immune response to infections

(Bhat et al 2010 Mendu et al 2011 Soltani et al 2011

Tian et al 2011 Wheeler et al 2011) bull Has even a role in Alzheimer disease stroke and

traumatic brain injury (Popovich and Longbrake 2008

Schwartz and Shechter 2010)

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Role of GABA ndash IN TYPE- I DM

bull Currently a trial with hypothesis that GABA a naturally occurring substance has the potential to reduce the inflammation and protect the pancreatic beta cells from autoimmune destruction

bull GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent onset Type I Diabetes

bull condition Type I Diabetesbull Drug Glutamic Acid Decarboxylase in alum formulation

bull Status -Phase 1

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

GABA amp SCHIZOPHRENIA

bull In subjects with schizophrenia impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC)

bull This dysfunction appears to be due at least in part to abnormalities in Gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry

bull Various experimental findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SSTNPY-containing and CCK containing subpopulations of GABA neurons and its signaling via certain GABA-A receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

GABA-B Receptor Complex Target for Tumor Therapy

A positive correlation between GABAB2 (minor b1)expression and that of thyroid tumors is seen

GABAB1 immunostaining of human ductal breast cancer tissues (from patents showsIntensive b unit staining in the cytosol and less frequently in the nucli

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

INTERVENTION CONDITION STATUS

1 GAD-Alum Juvenile onset type-1 DM

PHASE ndash 1 A

2 Merck L-830982GABA-A Alpha23 Receptor Agonist

Schizophrenia PHASE ndash 2

3 Pregabalin Pain after posteriar spinal fusion

PHASE - 4

4 Vigabatrin Coacaine abuse PHASE -2 A

5 Gabapentin Smoking PHASE ndash 1

6 Lomazenil ALCOHOL ABUSE PHASE -1

Trial scenario related to GABAhellip

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

Conclusionhellip

bull Despite the overwhelming representation of the

GPCRs in the human genome it is the ionotropic

receptors which achieved most visibility till today

The paucity of molecular heterogeneity exhibited by the

GABA-B receptors has proved problematic for specific drug targeting

bull Pharmacologists had to wait till gene knockout tecnique and readymade animal models became available till early 2000

bull The next decade will undoubtedly prove

Exciting with these recent genetic tools in hand

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120

THANK YOUhellip

• GABA RECEPTOR
• CONTENTS
• Neurotransmitter - GABA
• GABA SYNTHESIS UPTAKE AND METABOLISM
• GABA SYNTHESIS UPTAKE AND METABOLISM (2)
• ABChelliphellipTypes of GABA Receptor
• Subunits at GABA ndash A
• SUBUNITS OF GABA
• GABAA receptor structure
• Slide 10
• GABAA receptor MOA
• Video clip
• Extrasynaptic GABAA Receptors
• Extrasynaptic GABAA Receptors (2)
• Extrasynaptic GABAA Receptors amp drugs
• SUBUNIT AND PHARMACOLOGICAL ACTION
• GABAB - discovery
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• GABA-ρ subclass ( GABAC)
• Slide 23
• Slide 24
• PHARMACOLOGICAL APPLICATION OF GABA-A
• BASICS
• Slide 27
• Ibotenic acid and Muscimol
• GABOXADOL
• Slide 30
• Slide 31
• Slide 32
• Slide 33
• Slide 34
• Benzodiazepines (BDZ)
• Slide 36
• MOAhellip
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• NON BENZODIAZEPINE GABA MODULATORS
• ZALEPLON
• ZOLPIDEM
• ESZOPICLONE
• Inverse agonist at BZD Receptor
• Slide 47
• Slide 48
• FLUMAZENIL A BENZODIAZEPINE RECEPTOR ANTAGONIST
• FLUMAZENIL
• Barbiturates
• Antiepileptic actions at GABAA Receptors
• GABAergic terminal
• Inhibition of uptake increases GABA action
• VALPROIC ACID
• VALPROIC ACID (2)
• VIGABATRIN
• TIAGABIN
• GABAPENTIN amp PREGABALIN
• Slide 60
• Role OF GABA RECEPTORS IN ANAESTHESIA
• GENERAL ANAESTHETICS
• General anaesthetics
• Single amino acids determine iv anaesthetic activity
• A binding site for volatile anaesthetics
• Neuroactive steroids
• Slide 67
• NEUROACTIVE STEROIDS
• Slide 69
• Slide 70
• ALPHAXOLONE
• OTHER NEUROACTIVE STEROIDS
• Slide 73
• Alcohol
• NEUROSTEROID INVOLVEMENT IN THE GABAMIMETIC PROFILE OF ETH
• Slide 76
• Slide 77
• ROLE OF GABA IN ALCOHOL WITHDRAWAL
• GHB (Gamma hydroxy butyric acid)
• GHB (Gamma hydroxy butyric acid)
• EPIDEMIOLOGY OF GHB ABUSE
• Effectshellip
• Slide 83
• Slide 84
• BACLOFEN
• Baclofen
• Phenibut
• Phenibut (2)
• Phenibut (3)
• Slide 90
• Saclofen amp Phaclofen
• Slide 92
• Progabide
• Progabide (2)
• Picamilon
• Picamilon (2)
• GABA ndashC AGONIST
• TPMPA Selective GABA-C Antagonist
• GABA C
• Slide 100
• Section III Herbal Preperations and GABA Receptors
• Bicuculline
• Picrotoxin
• Muscimol
• Apigenin ndash GABA Receptor Modulator
• Epigallocatechin gallate (EGCG)
• Hispidulin and Related Flavonoids
• Section IV
• GAD 65 OR GAD 67
• GENE ORGANISATION OF SUBUNITS
• Slide 111
• Slide 112
• GABA ndash AS IMMUNOMODULATOR
• GABA ndash AS IMMUNOMODULATOR (2)
• Role of GABA ndash IN TYPE- I DM
• GABA amp SCHIZOPHRENIA
• GABA-B Receptor Complex Target for Tumor Therapy
• Slide 118
• Conclusionhellip
• Slide 120